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Biotechnology - Metabolic Engineering - Metabolic Flux Analysis

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Biotechnological Production of Alternate Sugars

1. Introduction:

The use of low-calorie sugar-free products tripled in the final two decades of the 20th century. In the United States alone, more than 150 million people use these products regularly. Even though hundreds of good-tasting low-calorie, sugar-free products are now available, most light product consumers say they would like to have additional low-calorie sugar-free products available. Of particular interest are baked goods and desserts. A number of events that occurred in the late 1990s are expected to facilitate providing additional good-tasting, low-calorie, sugar-free products. The approval of acesulfame potassium for soft drinks and aspartame and sucralose as general purpose sweeteners in the United States and recognition by regulatory agencies around the world that polyols have reduced caloric values compared with sucrose are examples. (A general purpose sweetener may be used in accordance with good manufacturing practices to sweeten any food when a standard of identity does not preclude its use.) These events should expand the use of sweeteners alone and in combination as well.

On the scientific front, after more than 100 years of use, scientists around the world are publicly acknowledging that saccharin is safe for humans. For example, in 1997, a special International Agency for Research on Cancer (IARC) panel determined the bladder tumors in male rats resulting from the ingestion of high doses of sodium saccharin are not relevant to man. And, in late 1998, IARC downgraded saccharin from a Category 2B substance, possible carcinogenic to humans, to Category 3, not classifiable as to its carcinogenicity to humans. Agents for which the evidence of carcinogenicity is inadequate in humans but sufficient in experimental animals may be placed in Group 3 when strong evidence exists that the mechanism of carcinogenicity in experimental animals does not operate in humans. This is the first time IARC has considered mechanistic data, and the IARC panel voted unanimously that saccharin could cause tumors in rats but that this is not predictive of human cancer.

Unfortunately, not all events surrounding sweeteners have been positive. Although some new technologies have provided a means of supporting the safety of sweeteners, the Internet has made disseminating negative information without accountability a new art form. No adverse health effects related to aspartame have been confirmed, but this has not stopped its critics. An extremely negative, inaccurate article making absurd claims about aspartame began circulating on the Internet in late 1998. The article asserts that aspartame is responsible for any number of ailments, without supporting data and creating new challenges for industry. Fortunately, many of the negative claims from this article and other antiaspartame fanatics are so absurd that the sources are not considered credible by many.

1.1. The Ideal Sweetener:

The search for the perfect sweetener continues, but it has long been recognized that the ideal sweetener does not exist. Even sucrose, the ‘‘gold standard,’’ is not perfect and is unsuitable for some pharmaceuticals and chewing gums.

Alternative sweeteners (a) provide and expand food and beverage choices to control caloric, carbohydrate, or specific sugar intake; (b) assist in weight maintenance or reduction; (c) aid in the management of diabetes; (d) assist in the control of dental caries; (e) enhance the usability of pharmaceuticals and cosmetics; (f) provide sweetness in times of sugar shortage; and (g) assist in the costeffective use of limited resources. The ideal sweetener should be at least as sweet as sucrose, colorless, odorless, and noncariogenic. It should have a clean, pleasant taste with immediate onset without lingering. The more a sweetener tastes and functions like sucrose the greater the consumer acceptability. If it can be processed much like sucrose with existing equipment, the more desirable it is to industry.

The ideal sweetener should be water soluble and stable in both acidic and basic conditions and over a wide range of temperatures. Length of stability and consequently the shelf-life of the final product are also important. The final food product should taste much like the traditional one. A sweetener must be compatible with a wide range of food ingredients because sweetness is but one component of complex flavor systems. Safety is essential. The sweetener must be nontoxic and metabolized normally or excreted unchanged, and studies verifying its safety should be in the public domain.

To be successful, a sweetener should be competitively priced with sucrose and other comparable sweeteners. It should be easily produced, stored, and transported.

1.2. Relative Sweetness:

Perceived sweetness is subjective and depends on or can be modified by a number of factors. The chemical and physical composition of the medium in which the sweetener is dispersed has an impact on the taste and intensity. The concentration of the sweetener, the temperature at which the product is consumed, pH, other ingredients in the product, and the sensitivity of the taster are all important. Again, sucrose is the usual standard. Intensity of the sweetness of a given substance in relation to sucrose is made on a weight basis.

1.3. The Multiple Ingredient Approach:

The advantages of the multiple sweetener approach have long been known. A variety of approved sweeteners are essential because no sweetener is perfect for all uses. With several available, each sweetener can be used in the application(s) for which it is best suited. Manufacturers also can overcome limitations of individual sweeteners by using them in blends.

During the 1960s, cyclamate and saccharin were blended together in a variety of popular diet soft drinks and other products. This was really the first practical application of the multiple sweetener approach. The primary advantage of this sweetener blend was that saccharin (300 times sweeter than sucrose) boosted the sweetening power of cyclamate (30 times sweeter), whereas cyclamate masked the aftertaste that some people associate with saccharin. The two sweeteners when combined have a synergistic effect—that is the sweetness of the combination is greater than the sum of the individual parts. This is true for most sweetener blends. Cyclamate was the major factor in launching the diet segment of the carbonated beverage industry. By the time it was banned in the United States in 1970, the products and trademarks had been well established.

Such a large market for diet beverages provided a tremendous incentive to develop new sweeteners. After cyclamate was taken off the market in 1970, saccharin was the only available low-calorie alternative to sugar available in the United States for more than a decade. But now with the availability of acesulfame potassium, aspartame, sucralose, and saccharin, the multiple sweetener approach is a visible reality in the United States. Fountain soft drinks generally contain a combination of saccharin and aspartame and bottled drinks are available with combinations of aspartame and acesulfame K, as well as sucralose and aspartame.

Triple blends, such as acesulfame potassium, aspartame, and saccharin and aspartame, cyclamate, and acesulfame potassium are being used in some parts of the world.

The polyols also are important adjuncts to sugar-free product development. These sweeteners provide the bulk of sugar but are generally less sweet than sucrose. The polyols, which are reduced in calories, combine well (e.g., they are synergistic) with low-calorie sweeteners, resulting in good-tasting reducedcalorie products that are similar to their traditional counterparts. With the availability of fat replacers and low-calorie bulking agents (e.g., polydextrose), not just a multiple sweetener approach but a multiple ingredient approach to calorie control is being used. In addition to the evidence that humans have an innate desire for sweets, research indicates that the obese and those who once were obese may have a greater preference than others for fatty liquids mixed with sugar. Replacing the fat and the sugar is therefore important in the development of products to assist in calorie control.

2. Alternate Sweeteners and their Productions:

A) High Intensity Sweeteners:

2.1. Acesulfame K:

Acesulfame K is 200 times sweeter than sucrose (table sugar), as sweet as aspartame, about 2/3 as sweet as saccharin, and 1/3 as sweet as sucralose. Like saccharin, it has a slightly bitter aftertaste, especially at high concentrations. Kraft Foods has patented the use of sodium ferulate to mask acesulfame's aftertaste. Acesulfame K is often blended with other sweeteners (usually sucralose or aspartame). These blends are reputed to give a more sugar-like taste whereby each sweetener masks the other's aftertaste, and/or exhibits a synergistic effect by which the blend is sweeter than its components.

Unlike aspartame, acesulfame K is stable under heat, even under moderately acidic or basic conditions, allowing it to be used as a food additive in baking, or in products that require a long shelf life. In carbonated drinks, it is almost always used in conjunction with another sweetener, such as aspartame or sucralose. It is also used as a sweetener in protein shakes and pharmaceutical products, especially chewable and liquid medications, where it can make the active ingredients more palatable.

2.1.1. Production:

Biotechnological production of Acesulfame K:

Not yet been identified.

2.2. Cyclamate:

Sodium cyclamate (sweetener code 952) is an artificial sweetener. It is 30–50 times sweeter than sugar (depending on concentration; it is not a linear relationship), making it the least potent of the commercially used artificial sweeteners. Some people find it to have an unpleasant aftertaste, but, in general, less so than saccharin or acesulfame potassium. It is often used with other artificial sweeteners, especially saccharin; the mixture of 10 parts cyclamate to 1 part saccharin is common and masks the off-tastes of both sweeteners. It is less expensive than most sweeteners, including sucralose, and is stable under heating.

2.2.1. Production:

Cyclamates are produced from cyclohexylamine (obtained by the reduction of aniline) by sulfonation. Production of sodium and calcium cyclamates in western Europe (annual capacity, 4200 tonnes) in 1995 was estimated to be 4000 tonnes. Brazil (annual capacities of 4000 and 2000 tonnes of sodium and calcium cyclamate, respectively) produced over 2300 and 300 tonnes of sodium and calcium cyclamate, respectively, in 1993. There is no commercial production of cyclamates in Japan or the United States. Information available in 1995 indicated that cyclamic acid was produced in Japan, Spain, Taiwan and the United States, that sodium cyclamate was produced in Argentina, Brazil, Germany, Indonesia, Japan, the Netherlands, Romania, South Africa, Spain, Taiwan, Thailand and the United States and that calcium cyclamate was produced in Argentina, Brazil, Japan, South Africa, Taiwan and the United States (Chemical Infor- mation Services, 1995). Sodium and calcium cyclamates are used as non-nutritive sweeteners; sodium cyclamate, simply known as ‘cyclamate’, is the more common salt. Calcium cyclamate is used in low-sodium and sodium-free products. World con- sumption of cyclamates in 1995 was estimated to be 15 000 tonnes. Western European con- sumption of cyclamates was estimated to be 4000 tonnes. Canadian consumption of cyclamates in 1995 was estimated to be 60 tonnes. Consumption of sodium cyclamate in China in 1995 was estimated to be 4400 tonnes.

Biotechnological production of Cyclamate:

Not yet been identified.

2.3. Alitame, Aspertame and Neotame:

Alitame is an artificial sweetener developed by Pfizer in the early 1980s and currently marketed in some countries under the brand name Aclame. Like aspartame, alitame is an aspartic acid-containing dipeptide. Most dipeptides are not sweet, but the unexpected discovery of aspartame in 1965 led to a search for similar compounds that shared its sweetness. Alitame is one such second-generation dipeptide sweetener. Neotame, developed by the owners of the NutraSweet brand, is another.

Alitame has several distinct advantages over aspartame. It is about 2000 times sweeter than sucrose, about 10 times sweeter than aspartame, and has no aftertaste. Its half-life under hot or acidic conditions is about twice as long as aspartame's, although some other artificial sweeteners, including saccharin and acesulfame potassium, are more stable yet. Unlike aspartame, alitame does not contain phenylalanine, and can therefore be used by people with phenylketonuria.

Aspartame is an artificial, non-saccharide sweetener used as a sugar substitute in some foods and beverages. In the European Union, it is codified as E951. Aspartame is a methyl ester of the aspartic acid/phenylalanine dipeptide. It was first sold under the brand name NutraSweet; It was first synthesized in 1965 and the patent expired in 1992.

The safety of aspartame has been the subject of several political and medical controversies, congressional hearings and Internet hoaxes since its initial approval for use in food products by the U.S. Food and Drug Administration (FDA) in 1981. A 2007 medical review on the subject concluded that "the weight of existing scientific evidence indicates that aspartame is safe at current levels of consumption as a non-nutritive sweetener". However, because its breakdown products include phenylalanine, aspartame must be avoided by people with the genetic condition phenylketonuria.

Neotame is an artificial sweetener made by NutraSweet that is between 7,000 and 13,000 times sweeter than sucrose (table sugar). In the European Union, it is known by the E number E961. It is moderately heat-stable, extremely potent, rapidly metabolized, completely eliminated and does not appear to accumulate in the body.

The major metabolic pathway is hydrolysis of the methyl ester by esterases that are present throughout the body, which yields de-esterified neotame and methanol. Because only trace amounts of neotame are needed to sweeten foods, the amount of methanol derived from neotame is much lower than that found in common foods.

The product is attractive to food manufacturers, as its use greatly lowers the cost of production compared to using sugar or high fructose corn syrup (due to the lower quantities needed to achieve the same sweetening), while also benefitting the consumer by providing fewer "empty" sugar calories and a lower impact on blood sugar.

2.3.1. Production:

Biotechnological Production:

All three above Sweeteners are amino acid and peptide based sweeteners, so they can be produced by formulation of individually produced Amino acids and peptides.

Production of amino acids by biotechnology methods Biotechnological production processes have been used for industrial production of amino acids for about 50 years now.

Microbiological (biotechnology) methods for the industrial production of amino acids are of three types: use of microbial enzymes or immobilized cells (enzymatic method), semi-fermentation, and direct fermentation. Enzyme catalysis is well established in the chemical industry. With the enzymatic method, an amino acid precursor is converted to the target amino acid using one or two enzymes. This method uses pure enzymes, rather than the enzyme systems of living microorganisms, as in the fermentation methods. The enzymatic method allows the conversion to a specific amino acid without microbial growth, thus eliminating the long process from glucose. Industrial use of enzymes for production of L-amino acids began about 40 years ago in Japan with the resolution of N-acetyl D,L-amino acids by immobilized acylase. Significant production by enzymatic catalysis is currently in place for alanine, aspartic acid, cysteine, cystine, methionine, phenylalanine, serine, tryptophan and valine. For example, L-aspartic acid is manufactured mainly by enzyme-catalyzed addition of ammonia to fumaric acid and this is the preferred manufacturing method. Since only the naturally occurring L-form is produced that way, resolution is not necessary, unlike for chemically synthesized amino acid racemic mixtures. For the production of L-methionine, enzymatic resolution with acylase from Aspergillus oryzae in the enzyme membrane reactor is the method of choice. Several hundred tons of L-methionine and L-valine are produced each year using this technology. L-cysteine is manufactured by industrially used enzymatic process in which the thiazoline derivative DL-2-amino- 2-thiazoline-4-carboxylic acid is converted with the help of three enzymes (L-ATC hydrolase, S-carbamoyl-Lcysteine hydrolase, and ATC racemase) from Pseudomonas thiazolinophilum.

According to Leuchtenberger et al. enzyme catalysis is a particularly elegant and popular method of producing D-amino acids and nonproteinogenic L-amino acids. The enzymatic production of L-tryptophan from precursors involves a single reaction step. It may be performed with isolated enzymes, either tryptophan synthase or tryptophanase, or by a variety of microorganisms with these enzyme activities such as E. coli. The enzymatic method has now been supplanted by a continuous microbiological process in which the reacting solution passes over a fixed bed of an immobilized microorganism. With semi-fermentation, a metabolic intermediate in the amino acid biosynthesis or its precursor is converted to the amino acid during fermentation. The fermentation method is being applied to industrial production of most L-amino acids. This method utilizes the phenomenon that microorganisms convert nutrients to various vital components they need. With the fermentation method, raw materials such as syrups are added to microorganism culture media, and the proliferating microorganisms are allowed to produce amino acids. Enzymes play an important role in the production of amino acids by fermentation. Consecutive reactions by 10 to 30 types of enzymes are involved in the process of fermentation, and various amino acids are produced as a result of these reactions. The fermentation may take place on a culture medium composed of grains, sugar, molasses, yeast, or other biological material, e.g. petrochemicals, such as paraffin, and synthetic nutrients such as ammonium chloride, ammonium nitrate, and potassium phosphate. Extraction is achieved by physical and mechanical means, such as heat or maceration, as well as by chemical methods such as petroleum solvents, ammonia, strong acids, strong bases, and/or ion exchange. The final product is obtained as crystalline powder. Once a suitable microorganism has been selected for the fermentation method, it is necessary to enhance its potential in order to take full advantage of the potential of the organism. Generally, microorganisms produce the 20 kinds of amino acids only in the amounts they need. They have a mechanism for regulating the quantities and qualities of enzymes to yield amino acids only in the amounts necessary for themselves. Releasing this regulatory mechanism allows manufacturing of the target amino acid in large amounts. The yield of an amino acid depends on the quantities and qualities of the enzymes. The yield increases if the enzymes involved in the production of the target amino acid are present in large quantities under workable conditions, whereas, if the enzymes are present in small quantities, it decreases. Strains are improved using various techniques.

Improvements in biotechnology production of amino acids:

The genome of the production organism is altered at random, and more efficient mutants are identified in a subsequent selection process. The mutants are usually characterized by increased membrane permeability, regulation defects, or biosynthesis enzymes with altered kinetic characteristics. Fundamental understanding of the microbial amino acid metabolism is necessary for further increases in productivity. Detailed analysis allows quantification of the flow of metabolites in a fermentation process as a function of time (metabolic flux analysis). In this way the addition of nutrients can be optimized, and yields can be increased as a result. Metabolic flux analysis also makes it possible to model the metabolism of a given production strain (metabolic modeling).

Another important requirement for the targeted improvement of amino acid production strains is knowledge about the genome of the microorganism concerned. The genome of C. glutamicum has already been sequenced. Not only fermentation processes, but also enzymatic processes are undergoing continual improvement, especially concerning the identification of novel enzymes. Unlike the classical screening, modern technologies access the genetic information directly. Specific characteristics of known enzyme systems can also be improved through incorporation of mutations into the genes of the biocatalyst. The optimized biocatalysts can also be transferred into production strains to avoid bottle necks in amino acid biosynthesis.

Direct production of Aspertame by rDNA technology:

Nonribosomal peptide synthesis is an alternative method for the production of pharmacologically and biotechnologically important products. Many microorganisms produce these as secondary metabolites by using nonribosomal peptide synthetases (NRPSs). The assembly of nonribosomally synthesized peptides is determined by the sequence of catalytically independent modules of the multifunctional NRPSs. Nonribosomal Asp-Phe formation. Hybrid dimodular peptide synthetases were generated by fusion of native gene fragments, to contain the domain sequence A(Asp)-PCP-C-A(Phe)-PCP-Te, and used for in vitro production of the dipeptide Asp-Phe.

Escherichia coli BL21(kDE3)-gsp was transformed with the plasmids which was incorporated with this Domain sequences. This E. coli strain carries the pREP4-gsp plasmid supplying the gene gsp coding for the 4¢-Ppant transferase Gsp. The cells were induced at D600 ¼ 0.6–0.7 with 0.2 mM isopropyl thio-b-D-galactoside and incubated for an additional 1.5 h at 30

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C before being harvested. Thus Aspertame will be directly produced by over expression of recombinant gene.

2.4. Neohesperidin Dihydrochalcone:

The genus Citrus is an important resource for natural products with direct benefits to humans ranging from health-benefiting compounds to flavor and aroma compounds used by the food, beverage, and cosmetics industries. Compounds of applied interest include flavonoids belonging to the flavanone subgroup, which accumulate to considerable levels in the leaves and fruit of citrus species where their biological role in planta is not clear. The bitter flavanone neohesperidin, abundant mainly in inedible citrus fruit species, is the substrate for the commercial production of neohesperidin dihydrochalcone (NHDC), a seminatural and generally regarded as safe lowcalorie sweetener with unique properties. NHDC is approximately 1500-fold sweeter than sucrose, is stable under heat and a wide pH range, and has also been documented to function as a bitterness blocker and flavor enhancer with diverse uses in the food, beverage, pharmaceutical, and animal feed industries. NHDC for human consumption is usually combined with other sweeteners resulting in (i) a synergistic sweetening effect allowing for lower overall concentrations of the sweetening compounds and (ii) masking of sweetener aftertastes to derive a flavor resembling sucrose. NHDC has been a commercial product for many years; however, production is limited by the availability of the substrate neohesperidin, which accumulates to significant levels only in inedible citrus species such as Citrus aurantium (bitter orange) and other citrus hybrids that are especially grown for this purpose. Neohesperidin has also been detected in Citrus aurantium callus cultures; however, the growth rate of citrus callus/cell cultures is extremely low, and therefore, this source does not have commercial potential. NHDC may also be produced by chemical conversion of naringin (the dominant bitter flavonoid in grapefruit peels) into neohesperidin, but the amount of grapefruit peels is limiting. An isomeric flavanone, the rutinoside hesperidin, is the predominant flavanone in orange peels (over 90%), as well as lemon and mandarin peels, and is readily available in large quantities as a byproduct of the vast orange juice industry. However, hesperidin (hesperetin-7-O-rutinoside) could not be converted into neohesperidin (hesperetin-7-O-neohesperidoside) by chemical or biological modification using the tools available up to now.

2.4.1. Production:

Biotechnological production:

Extraction of Hesperidin from Orange Peels and Hydrolysis.

Hesperidin was extracted from ripe orange (Citrus sinensis var. Shamouti) peels by a modification of a published protocol as follows: Orange peels (100 g) were pulverized in 500 mL of saturated Ca(OH)₂ using a homogenizer (the pH was measured to be _12). The homogenate was transferred to a bottle and flushed with nitrogen before closing. The homogenate was heated to 60 °C for 1 h and left to cool slowly, and the pH was adjusted to _12 by the addition of Ca(OH)₂. Peel particles were filtered out of the homogenate using gauze and filter paper. The homogenate was centrifuged at 20000g for 15 min, and the supernatant was collected and filtered through a nylon membrane (0.45 ím). The pH of the now clear solution was lowered to 3.5 and incubated at room temperature overnight to induce ring C closure and to precipitate the hesperidin. The precipitate was collected by centrifugation at 20000g for 15 min, and the supernatant was discarded. The precipitate was washed with 10 mL of acetone and dried into a powder.

For hydrolysis, 50 mg of hesperidin was dissolved in 500 µL of DMSO that was then added to 25 mL of preheated (65 °C) 0.25% aqueous H2SO4 and immediately autoclaved for two consecutive cycles at 121 °C for 25 min. Hydrolysis products were allowed to precipitate overnight at room temperature, the supernatant was discarded, and the precipitate was dried in a lyophilizer.

Process for Conversion of Orange Peel Hesperidin into Neohesperidin:

Process to convert hesperidin extracted from orange peels into neohesperidin using metabolic engineering and biotransformation via three steps: (i) extraction of hesperidin, (ii) full or partial hydrolysis of hesperidin sugar moieties, and (iii) biotransformation of hesperidin hydrolysis products into neohesperidin. (i) Various methods for the extraction of hesperidin from orange peels have been described and are appropriate for industrial applications. (ii) Hesperidin can be enzymatically hydrolyzed to hesperetin-7-O-glucoside (HG) and hesperetin using hesperidinase (a mixture of R-L-rhamnosidase and â-D-glucosidase); however, the solubility of hesperidin in aqueous solutions at temperatures compatible with enzymatic activity is very low, making this approach somewhat of a challenge for industrial applications.

Alternatively, efficient hydrolysis of hesperidin into HG and hesperetin can be achieved using dilute acid catalysis under high temperatures, which allows for efficient solubilization of hesperidin in the aqueous solution. This process is simple and is straightforward for application in the industry. (iii) A tobacco cell suspension culture containing endogenous 7-Oglucosyltransferase activity was transformed with the gene Cm1,2RhaT encoding a flavanone-7-O-glucoside-2-O-rhamnosyltransferase and was shown to convert naringenin and naringenin-7-O-glucoside into naringin. Because modifications on flavonoid ring B appear not to substantially affect the affinity of the enzyme to the flavonoid substrate, it is expected that HG is a substrate for the enzyme encoded by Cm 1,2RhaT. This expectation is further supported by the fact that the 1,2RhaT enzyme purified from pummelo leaves was active on both naringenin-7-O-glucoside and HG substrates. Therefore, we propose to use cell suspension cultures transformed with Cm 1,2RhaT for biotransformation of HG and hesperetin into neohesperidin in one or two enzymatic steps, respectively.

Conversion of neohesperidin into NHDC was first demonstrated by Horowitz and Gentili. This simple and established procedure isomerizes neohesperidin into a chalcone under alkaline conditions, and the reductive hydrogenation in the presence of a catalyst completes the conversion to NHDC.

2.5. Saccharin:

Saccharin is an artificial sweetener. The basic substance, benzoic sulfilimine, has effectively no food energy and is much sweeter than sucrose, but has a bitter or metallic aftertaste, especially at high concentrations. It is used to sweeten products such as drinks, candies, cookies, medicines, and toothpaste.

Saccharin is unstable when heated but it does not react chemically with other food ingredients. As such, it stores well. Blends of saccharin with other sweeteners are often used to compensate for each sweetener's weaknesses and faults. A 10:1 cyclamate : saccharin blend is common in countries where both these sweeteners are legal; in this blend, each sweetener masks the other's off-taste. Saccharin is often used together with aspartame in diet carbonated soft drinks, so that some sweetness remains should the fountain syrup be stored beyond aspartame's relatively short shelf-life. Saccharin is believed to be an important discovery, especially for diabetics, as it goes directly through the human digestive system without being digested. Although saccharin has no food energy, it may trigger the release of insulin in humans and rats, presumably as a result of its taste, but this is not conclusive as the same study states "No statistically significant changes in plasma insulin were found." 

2.5.1. Production:

A number of companies around the world manufacture saccharin. Most manufacturers use the basic synthetic route described by Remsen and Fahlberg in which toluene is treated with chlorosulfonic acid to produce ortho- and para-toluenesulfonyl chloride. Subsequent treatment with ammonia forms the corresponding toluenesulfonamides. ortho-Toluenesulfonamide is separated from the para-isomer (this separation is alternatively performed on the sulfonyl chlorides), and ortho-toluenesulfonamide is then oxidized to ortho-sulfamoylbenzoic acid, which on heating is cyclized to saccharin. ortho-Toluenesulfonamide can occur as a contaminant in saccharin produced by this process, but not in that produced by the Maumee process, described below. The only producer in the United States currently uses the Maumee process, in which saccharin is produced from purified methyl anthranilate, a substance occurring naturally in grapes. In this process, methyl anthranilate is first diazotized to form 2-carbomethoxybenzenediazonium chloride. Sulfonation followed by oxidation yields 2-carbomethoxybenzenesulfonyl chloride. Amidation of this sulfonyl chloride, followed by acidification, forms insoluble acid saccharin. Subsequent addition of sodium hydroxide or calcium hydroxide produces the soluble sodium or calcium salt.

China is the world’s largest producer of saccharin, accounting for 30–40% of world production, with an annual production of approximately 18 000 tonnes in recent years; its exports amounted to approximately 8000 tonnes. In 1995, the United States produced approximately 3400 tonnes of saccharin and its salts, and Japan produced approximately 1900 tonnes. In the past, several western European companies produced sodium saccharin; however, by 1995, western European production had nearly ceased due to increasing imports of lower-priced saccharin from Asia. Information available in 1995 indicated that saccharin was produced in 20 countries, calcium saccharin was produced in five countries, and sodium saccharin was produced in 22 countries.

Biotechnological Production:

Not yet identified.

2.6. Sucralose:

Sucralose is an artificial sweetener. The majority of ingested sucralose is not broken down by the body, so it is noncaloric. In the European Union, it is also known under the E number (additive code) E955. Sucralose is approximately 320 to 1,000 times as sweet as sucrose (table sugar), twice as sweet as saccharin, and three times as sweet as aspartame. It is stable under heat and over a broad range of pH conditions. Therefore, it can be used in baking or in products that require a longer shelf life. The commercial success of sucralose-based products stems from its favorable comparison to other low-calorie sweeteners in terms of taste, stability, and safety.

2.6.1. Production:

Sucralose is manufactured by the selective chlorination of sucrose (table sugar), which substitutes three of the hydroxyl groups with chlorine. This chlorination is achieved by selective protection of the primary alcohol groups followed by acetylation and then deprotection of the primary alcohol groups. Following an induced acetyl migration on one of the hydroxyl groups, the partially acetylated sugar is then chlorinated with a chlorinating agent such as phosphorus oxychloride, followed by removal of the acetyl groups to give sucralose.

Biotechnological production:

This procedure involves the chemical chlorination of raffinose to form a novel tetrachloroaffinose intermediate (6,4′,1″,6″-tetrachloro-6,4′,1″,6″-tetradeoxygalactoraffinose; TCR) followed by the enzymic hydrolysis of the α-1-6 glycosidic bond of TCR to give sucralose and 6-chlorogalactose. Synthesis of raffinose was achieved from saturated aqueous solutions of galactose and sucrose using a selected α-galactosidase from Aspergillus niger. When raffinose is used as a starting material for sucralose synthesis, this route has fewer steps than either the preceeding method using glucose-6-acetate as an intermediate or the complete chemical synthesis from sucrose.

B) Polyols: (Bulk Sweeteners):

Second category of sweeteners that can substitute for both the physical bulk and sweetness of sugar includes the sugar alcohols (also called “polyols”) sorbitol, mannitol, xylitol, isomalt, erythritol, lactitol, maltitol, hydrogenated starch hydrolysates, and hydrogenated glucose syrups and often termed as “sugar replacers” or “bulk sweeteners.” Other 2 sweeteners, namely, trehalose and tagatose, although are actually sugars rather than sugar alcohols yet are similar in function to the polyols. These sweeteners are being industrially explored for their application as food ingredient in food products in which the volume and texture of sugar, as well as its sweetness, are important, such as sugar-free candies, cookies, and chewing gum. Many of these products are marketed as “diabetic foods.” Some of these substances, such as sorbitol, xylitol, and tagatose, also occur naturally in certain fruits or other food while some are produced by yeast, fungi, and bacteria. Several health-promoting effects have also been attributed to the addition of these ingredients and hence addition/fortification to foods can lead to products with functional benefits. These sweeteners can either be directly added to foods, or use of these sweeteners-producing microorganisms can lead to natural foods containing these sweeteners.

Total global sugar alcohol production in the $10.92 billion global sweetener market has been estimated to be at 836905 tons. The consumption of sugar alcohols and high-intensity sugar is poised to rise as much as 15%. Sorbitol made up the largest share of sugar alcohols (more than 54%), and tagatose is emerging as another much sought after sweetener with a fast growth rate estimated at more than 20% to 25% in near future. Other sugar alcohols including erythritol, maltitol, and xylitol have also increased their share of this market.

Polyols and other bulk sweeteners have a couple of potential advantages over sugar as food ingredients :

Ø  Do not promote the development of dental caries (tooth decay) and hence, the USFDA has authorized a health claim pertaining to dental caries on foods made with polyols provided that these foods contain one or a combination of the approved sugar alcohols, meet the criteria for “sugar free,” and do not lower plaque pH in the mouth below 5.7 during consumption or up to 30 min afterward.

Ø  Produce a lower glycemic response than most sugars and starches do. Thus, their use may be advantageous for people with diabetes.

Ø  Have low calorific values due to their poor digestion and poor absorption.

2.7. Mannitol:

Mannitol, a naturally occurring polyol (sugar alcohol with 6 carbon atoms), is found in animals and plants. It is present in small quantities in most fruits and vegetables. Typically, it can be found in such plants as pumpkins, celery, onions, grasses, olives, mistletoe, and lichens. Mannitol is also found in manna, the dried exudate of the manna ash tree known as Fraxinus ornus. Manna is obtained by heating the bark of the tree and it can contain up to 50% mannitol. Hence, manna has been a commercial source of mannitol in Sicily, Italy. Marine algae, especially brown algae, are also rich in mannitol. Furthermore, mannitol is commonly found in the mycelium of various fungi and is present in fresh mushrooms (about 1%). Also, some fungi and bacteria produce mannitol. Moreover, small quantities of mannitol are found in wine. It is widely used in the food, pharmaceutical, medicine, and chemical industries. It is only half as sweet as sucrose. Mannitol also exhibits reduced caloric values compared to the respective value of most sugars, for example, the calorific values of mannitol and sucrose are 1.6 kcal/g and 4 kcal/g, respectively. The solubility of mannitol in water is significantly lower than that of sorbitol and most of the other sugar alcohols. At 14 °C, the solubility of mannitol in water is only approximately 13% while at 25 °C the solubility of mannitol in water is approximately 18%. Mannitol is sparingly soluble in organic solvents, like ethanol and glycerol, and practically insoluble in ether, ketones, and hydrocarbons. Mannitol forms white orthorhombic needles and the crystals have a melting point of 165 to 168 °C. A cooling sensation occurs when mannitol crystals dissolve in mouth. This effect is commercially used in chewing gums. Crystalline mannitol exhibits a very low hygroscopicity and is chemically inert. These properties make mannitol very useful in production of tablets and granulated powders. Mannitol is considered safe for use in foods and it has a food additive status. Mannitol is presently on the U.S. FDA GRAS-/INTERIM (generally recognized as safe) list.

Technological application

Mannitol is a valuable nutritive sweetener because it is nontoxic, nonhygroscopic in its crystalline form and has no teeth-decaying effects. At present, the main application for mannitol in the food industry is as a sweetener in sugar-free chewing gums and for dusting chewing gum sticks. In addition, mannitol is used as a bodying and texturizing agent, anticaking agent, and humectant. Mannitol has been shown to exhibit osmoprotecting effect during drying of LAB. Mannitol has the potential to extend the shelf life of various foodstuffs. As mannitol is only partially metabolized by humans and does not induce hyperglycemia, which makes it useful for diabetics and for so-called light foods. Besides, mannitol is commercially useful in making artificial resins and plasticizers.

Functional application

Mannitol belongs to a group of drugs referred to as osmotic diuretics. In medicine, mannitol (“Osmitrol”) is used to increase the formation of urine to prevent and treat acute renal failure, and also in removal of toxic substances from the body. Mannitol is also used to reduce both cerebral edema (increased brain water content) and intraocular pressure. Furthermore, it is used to alter the osmolarity of the glomerular filtrate in treating kidney failures. In the pharmaceutical industry, mannitol is commonly used as a constituent in chewable tablets and granulated powders. Furthermore, its sweet cool taste is used to mask the unpleasant taste of many drugs.

Mannitol has also been shown to have antioxidant effect by scavenging off free hydroxyl radicals. Kostopanagiotou studied the effect of mannitol in the prevention of lipid peroxidation during major liver resections performed during hepatic inflow occlusion. They concluded that mannitol has an antioxidant activity, but were unable to confirm a positive impact on the postoperative clinical course. Mendoza also found the antioxidant effect of mannitol on hyaluronan depolymerization. Hyaluronan (HA) was depolymerized by hydroxyl radicals generated from hydrogen peroxide and cupric ions. About 26.51 mM of mannitol was needed to decrease the degradation of HA by 50%.

2.7.1. Production:

Biotechnological Production:

Mannitol is a 6-carbon sugar alcohol that has been produced traditionally by chemical catalysis. However, yeast, fungi, and especially LAB are also known to produce mannitol. When compared fungal and bacterial processes for the production of mannitol, the latter proved to be better. Biotechnology has brought advances to the production in terms of substrate purity, process equipment requirements, and safety. Enzymatic methods have improved the yields and the use of microbes has brought versatility to the range of substrates that can be used in the processes. Some of the microbial processes are already industrially feasible and could be taken to further use.

Some homofermentative LAB were found to produce small amounts of mannitol intracellularly, for example, Streptococcus mutans, Lb. leichmanii, lactate dehydrogenase-negative mutant of Lb. plantarum, and lactate dehydrogenase deficient mutant of Lc. lactis.

The most promising production strategy reported so far has been the utilization of nongrowing cells of heterofermentative LAB for converting fructose to mannitol. Heterofermentative LAB belonging to the genera Lactobacillus and Leuconnostoc are the potent producer of mannitol. These bacteria contain the enzyme mannitol dehydrogenase to convert fructose into mannitol. The LAB reported to produce mannitol are Ln. pseudomesenteroides, Lactobacillus spp. B001, Lactobacillus spp. KY-107, Lactobacillus spp. Y-107 and Leuconostoc spp. Y-002, Ln. pseudomesenteroides ATCC 12291, Lb. sanfranciscensis, and most recently Saha studied mannitol production by Lb. intermedius NRRL B-3693 using inulin as a substrate. He reported that when fructose and inulin mixture (3: 5, total 400 g/L) was used as substrate, the bacterium produced 227.9 ± 1.8 g/L of mannitol. He also used molasses and corn steep liquor as a cheap substrate for mannitol production. Most studies on microbial mannitol production are based on batch cultivations and commonly only moderate production levels (yields or volumetric productivities) have been reported. The yield can be improved by screening for more efficient production strains. To improve the volumetric productivity of bioprocesses, the use of membrane cell-recycle bioreactor (MCRB) systems has been suggested. Soetaert and coauthors have studied extensively the production of mannitol with a heterofermentative lactic acid bacterium (LAB), Ln. pseudomesenteroides ATCC-12291. In fed–batch cultures an average volumetric mannitol productivity of about 6.3 g/L/h and a mannitol yield from fructose of 94 mol% ([mole of mannitol produced/mole of fructose used]× 100) were achieved. An improved volumetric productivity (8.9 g/L/h) but a low yield (60 mol%) was reported for cells immobilized to reticulated polyurethane foam. Ojamo suggested the production of mannitol by high densities of immobilized Ln. pseudomesenteroides cells. In this process, the average volumetric mannitol productivity and mannitol yield from fructose were approximately 20 g/L/h and 85 mol%, respectively. Weymarn studied the production of mannitol by heterofermentative LAB in a resting state. They developed a very efficient mannitol production process by combining membrane cell-recycle technology and high cell density batch cultures. They reported the volumetric mannitol productivity (26.2 g/L/h) and mannitol yield 97 mol%. Using the same initial biomass, a stable high-level production of mannitol was maintained for 14 successive bioconversion batches.

Helanto constructed random mutants of Ln. pseudomesenteroides by chemical mutagenesis for improving mannitol production of the mutant lacking the fructokinase activity. The best mutant showed fructokinase activity of only 10% of that of the wild-type strain. The effect of the random mutation on mannitol production was also studied in bioreactors under process conditions. The mutant strain grew and consumed sugars faster than the parent strain. Less side products (acids, carbon dioxide, and ethanol) were synthesized by the mutant cells and an increased fraction of fructose was reduced to mannitol by mannitol dehydrogenase (increased yield of mannitol from 74 to 86 mol%). The latest developments in the field have dealt with the use of recombinant strains in mannitol production.

In 2004, in response to a petition filed by zuChem Inc. (Chicago, Ill., U.S.A.), the USFDA amended additive regulations to permit the manufacture of mannitol by fermentation of sugars such as fructose, glucose, or maltose by the action of the microorganism Lb. intermedius (fermentum).

2.8. Tagatose:

Tagatose is an isomer of fructose that occurs naturally in some dairy products. In comparison to sugar it is metabolized differently, providing fewer calories and producing a smaller glycemic response. In chemical terms, D-tagatose has melting temperature, 134 °C; stable at pH 2 to 7; solubility, 58% (w/w) at 21 °C; viscosity, 180 cP at 70% (w/w) at 20 °C. It is involved in browning reactions during heat treatment and decomposes more readily than sucrose at high temperatures. Tagatose is derived from lactose, the sugar found in milk. During the production of tagatose, after enzyme hydrolysis of lactose the 2 monomers—galactose and glucose—are chromatographically separated. The galactose fraction is then converted to tagatose under alkaline conditions. Tagatose is a functional sweetener and is very similar in texture to sucrose (table sugar) and is 92% as sweet, but with only 38% of the calories.

D-tagatose is a malabsorbing sugar, as it is poorly absorbed in the small intestine. Its unabsorbed fraction is completely fermented by the intestinal microflora in intestine and the formed short-chain fatty acids are quickly absorbed and metabolized. During fermentation, there is a relatively low-energy recovery, and rather a certain amount of energy is lost due to increased biomass excretion of the microflora. D-tagatose has a sucrose-like taste with no cooling effect or aftertaste. It is similar to the polyols in having a low caloric value and tooth-friendly property. However, it has no laxative effect unlike polyols. Although similar to sucrose in taste, it does not contribute to calorie production. D-tagatose has been found not to contribute to net energy as revealed by a number of growth studies on rats and human clinical trials show that subjects gradually and consistently lose weight at medically desirable rates. As a result of these properties, D-tagatose is considered to be a potential reduced-energy sweetener.

Technological application

D-tagatose can be used as a low-calorie sweetener (1.5 Kcal), as an intermediate for synthesis of other optically active compounds, and as an additive in detergent, cosmetic, and pharmaceutical formulation. Besides effecting enhancement of flavor, it provides the natural taste and texture of sugar. According to Taylo who evaluated “Physical properties and consumer liking of cookies prepared by replacing sucrose with tagatose,” the latter appears to be suitable as a partial replacer for sucrose in cookies based on similar dough properties, cookie properties, and likeness scores. Using tagatose to replace sucrose in foods would reduce the amount of metabolizable sugars in the diet as well as provide the desirable prebiotic effect. On the commercial front, in 1996, Arla Foods Ingredients in the United States licensed the exclusive worldwide rights to produce and market tagatose for use in foods and beverages from Spherix. This sweetener is being marketed and sold as an ingredient under the Gaio name and used in chocolate products to be sold in New Zealand and Australia by Miada Sports Nutrition of New Zealand since 2004.

Functional application

A number of health benefits have been attributed to tagatose such as promotion of weight loss, no glycemic effect, antiplaque, noncariogenic, antihalitosis, prebiotic, and antibiofilm properties, organ transplants, improvement of pregnancy and fetal development, treatment of obesity, reduction in symptoms associated with type 2 diabetes, hyperglycemia, anemia, and hemophilia.

Prebiotic effect A small difference in chemical structure of tagatose compared to fructose gets translated into a significant difference in the overall metabolism of the sugar. The fructose carrier-mediated transport in the small intestine exhibits little affinity for tagatose, and only approximately 20% of ingested tagatose is absorbed in the small intestine. The absorbed part is metabolized in the liver a la fructose. The major part of ingested tagatose is fermented in the colon by the indigenous microflora resulting in the production of short-chain fatty acids. In this respect, tagatose is a potential prebiotic. Tagatose has been found to cause alteration in the composition of colonic microflora in pigs as evidenced by changes in the proportions of the short-chain fatty acids produced. This showed that the microflora favored by tagatose consumption had the potential to produce large amounts of butyrate, which is believed to be of importance for colonic health. Increased concentrations of butyrate were also observed in portal vein blood from both adapted pigs fed tagatose. The appearance of butyrate in the portal vein of unadapted pigs showed that adaptation to tagatose took place within the 12 h of the experimental period.

Similar trends were observed in humans consuming 3 × 10 g tagatose per day for 2 wk. In vitro fermentation of 1% tagatose with fecal samples from the human volunteers revealed that the proportion of butyrate was higher when the volunteers were adapted to tagatose (35 mol% after 4 h of incubation as compared to 25 mol% in samples from unadapted ones). The human fecal microflora also adapted to tagatose fermentation within the time span of the experiment (48 h).

Besides, tagatose effected modification in microbial population density in the feces of the human volunteers. Pathogenic bacteria (such as coliform bacteria) were reduced, and specific beneficial bacteria (such as lactobacilli and lactic acid bacteria) were increased. These results were in agreement with a study on evaluating pure strains of intestinal bacteria as well as dairy starter cultures for their ability to ferment tagatose. This study comprised isolates of normal (34 strains) or pathogenic (11 strains) human intestinal bacteria, 22 additional intestinal isolates from healthy humans, and 107 dairy-type lactic acid bacteria.

Tagatose was found only to be fermented by a limited number of the intestinal bacteria and except for one Clostridium species; the strains that were able to ferment tagatose belonged to the lactic acid bacteria group, Lactobacillus and Enterococus and pathogens failed to metabolize tagatose. The high frequency of tagatose fermentation between intestinal Lactobacillus and Enterococus was confirmed in the dairy-type lactobacilli and Enterococus species. None of the Bifidobacterium tested was able to ferment tagatose. Lactobacilli are important inhabitants of the intestinal tract of man and animals and they are believed to exert positive effects on intestinal function and health. As the viability of live bacteria in food products and during transit through the gastrointestinal tract may be variable, an alternative approach is to stimulate the growth of beneficial colonic bacteria by nondigestible food, the probiotic concept. The stimulation of lactobacilli and the increase in butyrate production in vitro and in vivo indicate that tagatose has prebiotic properties that could find important applications in functional foods.

Control of diabetes and obesity Early human studies suggested tagatose as a potential antidiabetic drug through its beneficial effects on postprandial hyperglycemia and hyperinsulinaemia. The incidence of diabetes is increasing worldwide representing a serious threat to public health. Glycemic control—that is, maintaining the blood glucose levels as close to the normal range as possible—has been shown to have positive effects against secondary complications such as retinopathy (The Diabetes Control and Complications Trial Research Group 1993. Intake of low glycemic foods, resulting in a lower blood glucose relative to a similar intake (50 g) of digestible carbohydrates from white bread or glucose, has been shown to improve glycemic control. In addition, avoiding obesity and increasing intakes of food rich in dietary fiber and low glycemic carbohydrate-containing foods have been advocated by WHO/FAO (1998) as the best means of reducing the rapidly increasing rates of type 2 diabetes. Similar types of foods have also been suggested to have beneficial effects against obesity and cardiovascular diseases. Lu carried out trials to confirm the potential of tagatose branded (Naturlose^®) for treating type 2 diabetes, and showed promise for inducing weight loss and raising high-density lipoprotein cholesterol, both important to the control of diabetes and constituting benefits independent of the disease. No current therapies for type 2 diabetes ensure these multiple health benefits. The predominant side effects of tagatose are gastrointestinal disturbances associated with excessive consumption, generally accommodated within 1- to 2-wk period. Under an FDA-affirmed protocol, Spherix is currently conducting a phase-3 trial to evaluate a placebo-subtracted treatment effect based on a decrease in HbA1c levels and possible side effects, and contraindications.

Antioxidant activity Tagatose has been observed to exhibit an antioxidant and a prebiotic, both properties cited in the maintenance and promotion of health. Paterna studied the antoioxidant properties of tagatose in cultured murine hepatocyte. They investigated the effects of tagatose on both the generation of superoxide anion radicals and the consequences of oxidative stress driven by prooxidant compounds in intact cells and observed that the extent of nitrofurantoin (redox cycling drug) (NFT) induced intracellular superoxide anion radical formation was not altered by tagatose. Consequently, they concluded that tagatose is a weak iron chelator, which can antagonize the iron-dependent toxic consequences of intracellular oxidative stress in hepatocytes. The antioxidant properties of tagatose may result from sequestering the redox-active iron, thereby protecting more critical targets from the damaging potential of hydroxyl radical.

It has been discovered that the addition of tagatose to an organ storage and preservative solution might reduce reperfusion injury of the organ during surgery and/or following removal of the organ from a subject. Tagatose exerts a dual effect that is beneficial in preserving the organ and preventing reperfusion injury. First, it is an aqueous-phase antioxidant. The mechanism of this protective effect against oxidative cell injury is iron chelation, sequestering iron from partitioning into membranes and promoting membrane lipid peroxidation. Second, exposure of organ cells such as liver cells to tagatose massively decreases ATP, which is beneficial in preventing apoptosis, as ATP is required for the apoptotic process to be initiated.

2.8.1. Production:

Biotechnological Production:

D-tagatose occurs naturally in Sterculia setigera gum, and it is also found in small quantities in various processed foods such as sterilized and powdered cow's milk, hot cocoa, and a variety of cheeses, yogurts, and other dairy products. D-tagatose can be produced from D-galactose by a chemical method using a calcium catalyst, but the process has some disadvantages, such as complex purification steps, chemical waste formation, and by-products formation. To overcome these limitations, biological manufactures of D-tagatose using several biocatalyst sources have been studied intensively in recent years. Among the biocatalysts, L-arabinose isomerase catalyzes the conversion of D-galactose to D-tagatose as well as the conversion of L-arabinose to L-ribulose, economically feasible tagatose manufacturing process. L-arabinose isomerase has been of interest for its potential application in galactose isomerization into tagatose and among other microorganisms; LAB were recognized as source of this enzyme. The LAB reported to contain L-arabinose isomerase are Lb. gayonii, Lb. plantarum, and Bifidobacterium longum. Cheetham invented a process to convert D-galactose to D-tagatose by using LAB. Further, Ibrahim have patented an enzymatic isomerization process using arabinose isomerase originating from a lactic acid bacterium. In a similar study, the L-arabinose isomerase from Lb. plantarum SK-2 was purified to an apparent homogeneity giving a single band on SDS–PAGE with a molecular mass of 59.6 kDa. The optimum activity observed at 50 °C and pH 7.0 was found further to be stimulated by Mn²⁺, Fe³⁺, Fe²⁺, and Ca²⁺ and inhibited by Cu²⁺, Ag⁺, Hg²⁺, and Pb²⁺. D-galactose and L-arabinose as substrates were isomerized with high activity. Using the purified L-arabinose isomerase, 390 mg tagatose could be converted from 1000 mg galactose in 96 h, and this production corresponds to 39% equilibrium.

The USFDA has granted GRAS status and authorized a health claim pertaining to tooth decay on products made with sugar alcohols to include tagatose as a substance eligible for the health claim, even though tagatose is not a sugar alcohol. Besides, tagatose is approved in Australia, New Zealand, Korea, and the European Union.

2.9. Sorbitol:

The most commonly used polyol in the United States is sorbitol, which is the standard sweetener in several sugar-free chewing gums and over-the-counter medicines. Sorbitol, also referred to as D-glucitol, is naturally found in many fruits, for example, berries, cherries, and apples and its worldwide production is estimated to be higher than 500000 tons/year and continues to rise. Sorbitol is sweet tasting, forms a viscous solution, stabilizes moisture, possesses bacterio-static property and is generally chemically inert. These features and properties make sorbitol an ideal and preferred ingredient in many products. It is freely soluble in water and acetic acid, ethanol, and methanol. It is insoluble in common organic solvent. Its melting point ranges from 93 to 98 °C. Sorbitol is often used in modern cosmetics as a humectant and thickener. Sorbitol is used as a cryo-protectant additive (mixed with sucrose and sodium polyphosphates) in the manufacture of surimi, a highly refined, uncooked fish paste most commonly produced from Alaska (or walleye) pollock (Theragra chalcogramma). Sorbitol, together with other polyhydric alcohols such as glycerol, is one of the ingredients in alkyl resins and rigid polyurethane foams manufacturing. In tobacco industries, sorbitol may give mild effect in sniff, good humectant agent, and is also to avoid acrolein formation which formed in burned glycerine. In textile industries, sorbitol is used as a softener and color stabilizer, and as a softener in leather industries.

Technological application

This polyol has a relative sweetness of around 60% vis-à-vis sucrose and displays a 20-fold higher solubility in water than mannitol. Owing these properties, sorbitol is widely used in a range of food products such as confectionery, chewing gum, candy, desserts, ice cream, and diabetic foods. In these products, it imparts sweetness and plays technological role such as a humectant, a texturizer, and a softener. Besides, sorbitol is the starting material for the production of pharmaceutical compounds such as sorbose and ascorbic acid. Several industrial processes have been described for the production of sorbitol.

Functional application

In medication, it is used as a laxative to treat occasional episodes of constipation. Vitamin C (ascorbic acid) is mainly semi-synthesized from sorbitol by fermentation process by Baccillus suboxydant. In oral or topical preparation, it is used as humectant, sweetener, bodying and viscosity agent, vehicle, anticaplocking, and texture improvement. In other case, sorbitol is useful to promote the absorption of certain minerals such as Cs, Sr, F, and vitamins B12. In high concentration, sorbitol is a stabilizer for unstable vitamins and antibiotics. It is used as an excipient and intravenous osmotic diuretic in pharmaceutical fields (PT. Sorini Corp., Tbk, Jawa Timur, Indonesia).

Sorbitol also has antioxidant properties. Shih-Yung studied the differential effect of sorbitol and polyethylene glycol on antioxidant enzymes in rice leaves. They reported that sorbitol treatment had no effect on lipid peroxidation; however, there is an increase in peroxidase, ascorbate peroxidase, and glutathione reductase activities in rice leaves treated with sorbitol. Findings led them to suggest that sorbitol treatment can upregulate antioxidant system in rice leaves.

2.9.1 Production:

Biotechnological Production:

Sorbitol is claimed to have important health-promoting effects. A recombinant strain of Lb. casei was constructed, cells of which when pre-grown on lactose, were able to synthesize sorbitol from glucose. Inactivation of the L-lactate dehydrogenase gene led to an increase in sorbitol production. Lb. casei is a lactic acid bacterium relevant as probiotic and used as a cheese starter culture. A sorbitol-producing Lb. casei strain might therefore be of considerable interest in the food industry.

Ladero reported the capacity of Lb. plantarum, a lactic acid bacterium found in many fermented food products and in the gastrointestinal tract of mammals, to produce sorbitol from fructose-6-phosphate by reverting the sorbitol catabolic pathway by over expressing sorbitol 6-P-dehydrogenase and the mutant strains deficient for both L- and D-lactate dehydrogenase activities.

2.10. Trehalose

Trehalose, also known as mycose, is a natural alpha-linked disaccharide formed by an α, α-1, 1-glucoside bond between 2 α-glucose units. In 1832, Wiggers discovered trehalose in an ergot of rye and in 1859 Berthelot isolated it from trehala manna, a substance made by weevils, and named it trehalose. Trehalose is found naturally in insects, plants, fungi, and bacteria; the major natural dietary source is mushrooms. It is implicated in anhydrobiosis—the ability of plants and animals to withstand prolonged periods of desiccation. It has high water retention capabilities and is used in food and cosmetics. The sugar forms a gel phase as cells dehydrate, which prevents disruption of internal cell organelles by effectively splinting them in position. Rehydration then allows normal cellular activity to be resumed without the major, lethal damage that would normally follow a dehydration/rehydration cycle. Trehalose has the added advantage of being an antioxidant. Trehalose is a naturally occurring reducer of cell stress, protecting these organisms from extremes in heat shock and osmotic stress. It acts by altering or replacing the water shell that surrounds lipid and protein macromolecules. It is thought that its flexible glycosidic bond allows trehalose to conform to the irregular polar groups of marcromolecules. In doing so, it is able to maintain the 3-dimensional structure of these biologic molecules under stress, preserving biologic function. Trehalolipids (trehalose linked at C-6 and C-6′ to mycolic acid) is produced from Rhodococcus erythropolis and Arthrobacter spp. acts as biosurfactant.

Trehalose has been accepted as a novel food ingredient under the GRAS terms in the United States and European Union. Trehalose has also found commercial application as a food ingredient. The uses for trehalose span a broad spectrum that cannot be found in other sugars, the primary one being its use in the processing of foods. Trehalose is used in a variety of processed foods such as dinners, western and Japanese confectionery, bread, vegetables side dishes, animal-derived deli foods, pouch-packed foods, frozen foods, and beverages, as well as foods for lunches, eating out, or prepared at home. Technology for the production of trehalose was developed in Japan, where enzyme-based processes convert wheat and corn syrups to trehalose. It is also used as a protein-stabilizing agent in research. It is particularly effective when combined with phosphate ions. Trehalose has also been used in several biopharmaceutical monoclonal antibody formulations: trastuzumab, marketed as Herceptin by Genentech, and ranibizumab, marketed as Lucentis by Genentech and Novartis. Because of its moisture-retaining capacity, it is used as a moisturizer in many basic toiletries such as bath oils and hair growth tonics. Using trehalose's properties to preserve tissue and protein to full advantage, it is used in organ protection solutions for organ transplants. Other fields of use for trehalose span a broad spectrum including fabrics that have deodorization qualities, plant activation, antibacterial sheets, and nutrients for larvae.

Technological application

It is anticariogenic, synthesized by several microorganisms, only partially digested by humans, and therefore considered a dietetic sugar. The protection of proteins against denaturation under stress conditions is a well-known property of trehalose. Trehalose is used in a wide range of products due to the multifaceted effects of trehalose's, such as its inherently mild, sweet flavor; its preservative properties that maintain the quality of the 3 main nutrients (carbohydrates, proteins, fats); its powerful water-retention properties that preserve the texture of foods by protecting them from drying out or freezing; and its ability to suppress bitterness, stringency, harsh flavors, and the stench of raw foods, meats, and packaged foods.

Functional application

Several recent studies have revealed health-promoting attributes of trehalose. In rats, it was shown to almost completely suppress dental caries, and in humans it reduced significantly acidification in plaques. Furthermore, it has been demonstrated that trehalose has important stabilizing effects on human proteins, preventing protein aggregation as well as formation of pathological con formational forms. As an extension of its natural capability to protect biological structures, trehalose has been used for the preservation and protection of biologic materials. It stabilizes bioactive-soluble proteins such as monoclonal antibodies and enzymes for medical use. It stabilizes proteins for inhaled use. It has been successfully used to preserve embryos, and cellular blood products. Cyopreservation of transplant cells and tissue in the presence of trehalose has been shown to increase viability and decrease host immune response.

As it inhibits lipid and protein misfolding, trehalose has become an attractive molecule for study in neurodegenerative disease characterized by protein misfolding and aggregate pathology. Such diseases include Alzheimer's and Parkinson's disease, and the less common triplet repeat diseases. Recent scientific publications describe trehalose benefit in model systems that recapitulate aggregate pathology that characterize Alzheimer's (AD), Huntington's (HD), and occulopharyngeal muscular dystrophy (OPMD). Having beneficial properties, its production in food products at the expense of other sugars is desirable.

Luo examined the effects of trehalose on the activities of key antioxidant enzymes, including superoxide dismutases (SODs), ascorbate catalases (CATs), and ascorbate peroxidases (APX) from wheat (Triticum aestivum), and then measured the ability of trehalose to scavenge hydrogen peroxide (H₂O₂) and superoxide anions (O₂-•). They indicated that trehalose protected SOD activity slightly. However, it inhibited CAT and APX activities under heat stress, with a little protection of CAT activity and trehalose scavenged H₂O₂ and O₂-• greatly in a concentration-dependent manner. Their results suggest that trehalose plays a direct role in eliminating H₂O₂ and O₂-• in wheat under heat stress. Kazuyuki studied the inhibitory effect of trehalose on the autoxidation of unsaturated fatty acids (UFA) by water/ethanol system and reported remarkable inhibition of formation of hydroperoxide from linoleic acid by trehalose. They also observed that the inhibitory effect on the autoxidation was dependent on the amount of trehalose. Similar to linoleic acid, the formation of hydroperoxide from α-linolenic acid was inhibited by trehalose. On the other hand, when the degradation of hydroperoxide was tested under the same conditions of the autoxidation, it was found that the degradation was not influenced by the presence of trehalose. Thus, it is clear that trehalose inhibits the autoxidation of unsaturated fatty acids, but negligibly suppresses the generation of volatile aldehydes from hydroperoxides. Trehalose depresses the effect on the oxidation of UFA through the weak interaction with the double bond(s).

2.10.1. Production:

Biotechnological Production:

Trehalose is widespread within the genus Propionibacterium. Trehalose accumulation in Propionibacterium, for example, P. acidipropionici and P. freudenreichii subsp. shermanii has also been observed to occur in response to stress conditions. In particular, P. freudenreichii subsp. shermanii strain NIZO B365 accumulates trehalose to remarkable levels, and the trehalose content increases considerably in response to osmotic, oxidative and acid stress (up to 40% [w/w] of the cell protein). In this organism, trehalose results from the conversion of glucose 6-P and ADP glucose via trehalose 6-P synthase to trehalose 6-P and its subsequent dephosphorylation by trehalose 6-P phosphatase. Alternatively, trehalose can be formed from maltose through the action of trehalose synthase.

While working on isolates of Propionibacteria from Indian fermented milks for vitamin B12 production in our laboratory, we have also incidentally observed trehalose production by these isolates as estimated by the HPLC method.

The safety of trehalose is supported by animal and human studies and is marketed in the United States as approved by USFDA since 2000. Trehalose is also approved in Japan, Korea, Taiwan, and the United Kingdom.

2.11. Erythritol:

Erythritol is a biological sweetener with applications in food and pharmaceuticals. Not only animal toxicological but also clinical studies have consistently demonstrated its safety even when consumed on a daily basis in high amounts. Large-scale production of erythritol uses fermentative processes with pure glucose, sucrose and dextrose from chemically and enzymatically hydrolyzed wheat and corn starches used as major carbon sources (Aoki et al. 1993; Marina et al. 1993). Erythritol is produced by fermentation involving yeast-like fungi such as Trigonopsis variabilis, Trichosporon sp., Torula sp., Candida magnoliae and Moniliella sp. Leuconostoc oenos can also produce erythritol but only under anaerobic conditions. A high initial concentration of glucose favors erythritol production by osmophilic microorganisms. Generally, an increase in the initial glucose concentration increases the production rate and yield in a batch process if the microorganisms can tolerate a higher concentration of sugar and a higher osmotic pressure. Erythritol has been produced commercially using a mutant of Aureobasidium. The mutant produced erythritol at 1.8 g l-1 h-1 with a 44% yield in a medium containing 40% (w/v) glucose. Although erythritol fermentation in synthetic media containing glucose or fructose has extensively been studied, little information is available about the production of this compound by Yarrowia lipolytica from glycerol. New uses of glycerol and new microbial transformations to interesting products are of increasing importance. What seems to be important for an economically competitive fermentation process is the ability of Y. lipolytica to grow and to produce erythritol on renewable products from industrial processes (such as raw glycerol discharged after biodiesel manufacture), which are low-cost carbon substrates. A cost reduction in erythritol production can be achieved by using less expensive substrates. We have recently reported that an acetate-negative mutant of Y. lipolytica Wratislavia K1 has the ability of simultaneously producing high amounts of erythritol and citric acid in media containing glycerol, at pH 5.5 which is optimal for citric acid biosynthesis by Y. lipolytica. In the current study, we evaluate the dynamics and yield of erythritol production from raw glycerol by the strain Wratislavia K1 at various pH, and thereafter investigate, in fed-batch experiments, the effect of the optimal pH on the production of erythritol by other strains belonging to the species Y. lipolytica.

2.11.1. Production:

Biotechnological Production:

Microorganisms and media

The following strains were used in this study: three acetate negative mutants of Yarrowia lipolytica: Wratislavia AWG7, Wratislavia 1.31 and Wratislavia K1, one mutant of Y. lipolytica 8661 UV1, and two wild strains of Y. lipolytica: A-101 and 1.22. All were from Wroclaw University of Environmental and Life Sciences, Poland. The growth medium for inoculum preparation contained: 50 g glycerol l-1, 3 g yeast extract l-1, 3 gmalt extract l-1 and 5 gBactopepton l-1. Erythritol production was conducted in a medium consisting of 150 g glycerol l-1, 3 g NH4Cl l-1, 1 g MgSO47H2O l-1, 0.2 g KH2PO4 l-1, and 1 g yeast extract l-1. After 48 h of cultivation, raw glycerol solution (86%w/v) was added (at a constant feeding rate of 1.4 g h-1) until a total of 300 g glycerol l-1 was obtained.

Culture conditions

A seed culture was grown in a 300 ml flask (containing 50 ml of growth medium) on a shaker at 30C for 3 days. An inoculum of 200 ml was introduced into the fermenter, which contained 1.8 l of the production medium. All fed-batch cultures were performed in a 5 l jar fermenter (Biostat B Plus, Sartorius, Germany) with a working volume of 2 l at 30C for 7 days. The aeration rate was 0.6 l min-1. The stirrer speed was adjusted to 800 rpm and the pH was maintained automatically at 3 by the addition of NaOH (20% w/v). All the cultures were conducted in two replications.

2.12. Isomalt:

Isomalt is an odourless, white, crystalline substance containing about 5 % water of crystallisation. It tastes just as natural as sugar, is not sticky, toothfriendly (it helps to prevent cavities and plaque), suitable for diabetics and has only about half as many calories as sugar because it cannot be completely metabolized. Isomalt is a bulk sweetener and can substitute sugar in a 1:1 mass ratio. It should not be mistaken for intense sweeteners, which have much greater sweetening power (between a hundred and a thousand times as high). For this reason these intense sweeteners are only used in small quantities. Because isomalt is similar to sucrose, it is particularly suitable for making products such as candies, chewing gum, chocolate, compressed tablets or lozenges, baked goods, baking mixtures, and pharmaceutical products using conventional equipment. Isomalt’s nutritional and physiological benefits are ideal for use in sugarfree, lowcalorie and diabetic products. It is particularly suitable for food and pharmaceutical applications and also offers many advantages for technical uses.

2.12.1. Production:

Biotechnological Production:

The microorganism Leuconostoc mesenteroides was found to produce an unknown disaccharide in1952. In 1957 Weidenhagen and Lorenz found independently that a bacterium they had isolated from sugar beet raw juice effects a conversion of sucrose to an unknown reducing disaccharide. The disaccharide was identified as isomaltulose and given the trivial name palatinose. It was identical with the unknown disaccharide Stodala, Koepsell, and Sharpe had isolated in 1952. The name palatinose is derived from „palatinum“, the latin name of the German province where Weidenhagen and Lorenz found the disaccharide. In 1958 Windisch identified the isolated bacterium as Protaminobacter rubrum den Dooren de Jong. Depending on culture conditions, P. rubrum produces a red pigment. The bacterium is also able to degrade and modify amines. But its most remarkable property is the transglycosylation of sucrose to palatinose. The first patent for the production of palatinose was awarded to Süddeutsche Zucker AG, Mannheim, Germany in 1957. In addition to P. rubrum the microorganisms Leuconostoc mesenteroides, Serratia plymuthica, Serratia marcescens, Erwinia carotovera, and Erwinia rhapontici have been reported to be capable of transforming sucrose into palatinose. The enzymatic transglycosylation of sucrose to palatinose effected by the glycosyltransferase (sucrosemutase) is described with the molecules in their steric representation in figure 2 for a better understanding. The glucosyl group of the sucrose (alpha-Dglucopyranosyl- 1,2-beta-D-fructofuranoside) changes from alpha(1->2) to alpha(1->6) position. The enzymatic reaction results in the thermodynamically more stable palatinose (alpha-glucopyranosyl-1,6-D-fructofuranose). Of all the microorganisms reported, only the strain of P. rubrum originally isolated by Weidenhagen and Lorenz in 1957 has been foreseen for industrial scale use thus far.

Earlier processes using viable, free microorganisms have the disadvantage of higher product purification costs and lower yield. More cost-effective are techniques using immobilized non-viable cells. This is in agreement with many other authors who prefer the use of immobilization techniques for other processes. The process starts with the propagation of the cells. P. rubrum can be cultivated either on thick juice supplemented with corn steep liquor or on molasses with additional nitrogen and phosphate e. g. (NH4)2HPO4. Molasses is a byproduct of the sugar industry. The growth medium is adjusted to a concentration of 5 % total solids and pH of 7.2. Sterilization of the medium is, of course, necessary.

In 1990, a large scale plant for production of isomalt went on stream in Offstein/ Rhineland-Palatinate. Current isomalt production is about 35,000 tons yearly. Hydrogenation of palatinose solutions can be done easily under moderate temperature and pressure conditions and at neutral pH or under mildly alkaline conditions. Any of the usual hydrogenation catalysts is suitable for carrying out the reaction. Quite suitable is Raney-Nickel in pelletized form in a fixed bed reactor. Theoretically the reduction of palatinose should yield an equimolar mixture of 1-O-alpha-Dglucopyranosyl- D-mannitol (1,1-GPM) and 6-Oalpha- D-glucopyranosyl-D-sorbitol (1,6-GPS) but depending on the conditions of hydrogenation the rate of each component can vary between 43-57 %.

2.13. Xylitol:

Xylitol is a five-carbon sugar alcohol that is naturally found in some fruits and vegetables. The most significant application of xylitol is its use as an ideal sweetener for diabetic patients. Other potential uses of xylitol are: as an anticariogenic agent in toothpaste formulations, as thin coatings on vitamin tablets, in chewing gum, soft drinks, mouthwashes, beverages, and in bakery products. Xylitol has received global demand mainly due to its insulin-independent metabolism, anticariogenecity, high sweetening power, and pharmacological properties. Xylitol is currently approved for usage in foods, pharmaceuticals, and oral health products in more than 50 countries. Xylitol can be produced either by chemical hydrogenation of pure xylose or by biotechnological processes. Currently, xylitol is industrially produced by chemical reduction of pure xylose in the presence of a nickel catalyst at high temperature and pressure. The yield of xylitol is about 50–60% of the xylan fraction and the resultant product is very expensive due to extensive purification procedures. The chemical process is laborious, and cost- and energy-intensive. In view of alternatives to the conventional process, two biotechnological approaches seem promising: the fermentation process and the enzymatic approach. These biotechnological processes are highly attractive alternatives that are able to produce a high-quality and cost-effective product. The fermentation process uses bacteria, fungi, and yeast for xylitol production from xylose or hemicellulosic hydrolysate. Yeasts are considered as the best xylitol producers among the microorganisms investigated. In the fermentation process, the yield of xylitol obtainable from xylose is in a range of 65–85% of the theoretical value (Nigam and Singh, 1995). The application of the fermentation process on an industrial-scale is time-consuming because of some preparatory activities such as sterilization and regular inoculum development. An advantage of the fermentation process over chemical procedures is its lower cost due to the non-necessity of xylose purification. However, the fermentation method has not yet been able to accumulate the advantages of the chemical process. Xylitol production from xylose using enzyme technology can be an attractive alternative to both fermentation and chemical processes. Compared to the fermentation process, the enzymatic approach employing xylose reductase (XR) for xylitol synthesis is expected to obtain a substantial increase in productivity. There are scarce reports on the enzymatic conversion of synthetic xylose to xylitol using XR. The conversion of D-xylose to xylitol is more than 95% by the NADH-dependent XR from yeast. Despite a wide range of applications, the use of xylitol as sweetener is limited due to its high price. This has inspired researchers to work toward the development of improved technologies to lower the production costs. In this field, the enzymatic approach to xylitol production from xylose present in the lignocellulosic biomass may provide an alternative for the chemical process. This review attempts to describe the current literature on the processes involving xylitol production, taking into account the chemical and biotechnological processes, microorganisms involved and tries to identify ways to improve enzymatic xylitol production so that it can compete with the chemical process.

2.13.1. Production:

Chemical Production:

Xylitol is manufactured industrially by reducing pure xylose, obtained from hardwood or hemicellulosic hydrolysate in the presence of a Raney nickel catalyst. The chemical synthesis of xylitol starts with the extraction of xylose from hemicellulose by acid-catalyzed hydrolysis. After color removal and purification, xylose-rich hemicellulosic hydrolysate can be employed for xylitol production through hydrogenation of xylose at 80–140°C and hydrogen pressures up to 50 atmospheres in the presence of metal catalysts (Raney nickel). The xylitol solution formed requires further purification by chromatography, and then concentration and crystallization of the product to obtain pure xylitol. The xylitol yield is only about 50–60% of the xylan fraction and thus the xylitol production process is expensive due to the extensive separation and purification stages.

Biotechnological Production:

Fermentation process

The fermentation process uses bacteria, fungi, and yeast for xylitol production from commercial pure xylose or hemicellulosic hydrolysate. The production of xylitol using bacteria and fungi has been studied to a lesser extent compared to that using yeast strains. A few bacteria, such as Enterobacter liquefaciens, Corynebacteriurn sp., and Gluconobacter oxydans, have been reported to produce xylitol. There are very few studies regarding xylitol production from D-xylose using filamentous fungi. Yeasts are considered as the best xylitol producers among the microorganisms. As a result, yeasts have been studied extensively in the last few decades by several researchers. Forty-four yeast strains from the five genera were screened by Barbosa for their ability to convert D-xylose to xylitol. Candida guilliermondii and C. tropicalis were found to be the best xylitol producers and these yeasts produced 77.2 g l-1 xylitol from 104 g l-1 xylose using high cell densities and a defined medium under aerobic conditions. The fermentation conditions were optimized by da Silva and Afschar (1994) during continuous cultivation of Candida tropicalis for xylitol production. C. tropicalis produced xylitol at a yield of 77–80% of theoretical value (0.91 g g-1) in a medium containing 100 g l-1 D-xylose. The screening of different xylose-assimilating yeast has confirmed that the best xylitol producers belong to the genus Candida. In the fermentation process using yeast, the yield of xylitol obtainable from D-xylose is in a range of 65–85% of the theoretical value. The production of xylitol through the fermentation process is limited by certain factors, such as precise control of culture conditions, expensive nutrients, huge water consumption, and the type of process. Thus, the application of the fermentation process on an industrial level is timeconsuming, being associated with some preparatory activities such as sterilization and regular inoculum development involving input of energy, labor, and time, leading to decreased productivity. The advantage of the fermentation process over chemical procedures is its lower cost due to the non-necessity of extensive xylose purification. The fermentative xylitol production has been studied as an alternative, but its viability is dependent on the optimization of the various fermentation variables such as nutritional composition (substrate, nitrogen source, and micronutrients), the culture and process conditions, as well as the genetic nature of the microorganisms.

Enzymatic process

The production of xylitol from xylose by using enzyme technology is an alternative and promising approach. The enzymatic conversion of D-xylose into xylitol using xylose reductase (XR) of Candida pelliculosa coupled with the oxidoreductase system of Methanobacterium sp. has been reported by Kitpreechavanich. The authors observed that the xylose was stoichiometrically converted to xylitol with an equivalent consumption of NADPH and that an almost quantitative conversion of xylose to xylitol was achieved using a NADP+-toxylose ratio of over 1:30, whereas the coenzyme was successfully regenerated and retained using a membrane reactor. About 90% conversion of xylose to xylitol could be achieved at 35°C and pH 7.5 after a 24 h reaction period. Nidetzky optimized the production of xylitol from xylose by XR from Candida tenuis coupled with glucose dehydrogenase from Bacillus cereus for regenerating the NADH in an enzyme reactor. In this system, the substrate was converted at concentrations of 300 g l-1 xylose, with a 96% yield and xylitol productivity of 3.33 g l-1 h-1. Neuhauser et al. (1998) reported on the C. tenuis XR-mediated NADH-dependent xylose reduction coupled with formate dehydrogenase (FDH) from C. boidinii for the byproduct-free recycling of NADH used in a pH-controlled enzyme reactor. In this process, a fed-batch conversion of 0.5 M xylose to xylitol using yeast XR yielded productivities of 2.8 g l-1 h-1. To optimize the performance of the XRcatalyzed reactions for xylitol synthesis, the effect of several process variables on productivity needs to be studied: pH, temperature, initial substrate, and coenzyme concentration.

Limitations:

Despite the yield of microbiological conversion of xylose to xylitol could be increased by 65–85% using different production methods, the chemical process would still be very competitive in terms of industrial scale manufacture. The synthesis of xylitol from xylose using XR from yeast is an attractive alternative to chemical and microbial processes. The application of XR constitutes an alternative of economic interest regarding both the chemical reduction of pure xylose and the fermentation of xylose present in the hemicellulosic hydrolysate. During enzymatic production, all experiments were performed using commercial xylose-containing medium. It is certainly still necessary to study the enzymatic conversion of xylose in the lignocellulosic biomass to xylitol and to optimize the reaction conditions. The production of xylitol through the chemical process is expensive due to difficult separation and purification steps. On the other hand, the fermentation process on an industrialscale is not feasible due to reduced productivity. Hence, it is important to explore alternative methods for the effective production of xylitol using XR enzyme. The enzymatic approach might be able to overcome the disadvantages of the chemical process that is largely being used at present and also the fermentation process that is under investigation.