Technological and Functional Applications of Low-Calorie Sweeteners from Lactic Acid Bacteria
ABSTRACT: Lactic acid bacteria (LAB) have been extensively used for centuries as starter cultures to carry out food fermentations and are looked upon as burgeoning “cell factories” for production of host of functional biomolecules and food ingredients. Low-calorie sugars have been a recent addition and have attracted a great deal of interest of researchers, manufacturers, and consumers for varied reasons. These sweeteners also getting popularized as low-carb sugars have been granted generally recommended as safe (GRAS) status by the U.S. Federal Drug Administration (USFDA) and include both sugars and sugar alcohols (polyols) which in addition to their technological attributes (sugar replacer, bulking agent, texturiser, humectant, cryoprotectant) have been observed to exert a number of health benefits (low calories, low glycemic index, anticariogenic, osmotic diuretics, obesity control, prebiotic). Some of these sweeteners successfully produced by lactic acid bacteria include mannitol, sorbitol, tagatose, and trehalose and there is a potential to further enhance their production with the help of metabolic engineering. These safe sweeteners can be exploited as vital food ingredients for development of low-calorie foods with added functional values especially for children, diabetic patients, and weight watchers.
Lactic acid bacteria (LAB) have been used since dawn of civilization for preparation of fermented foods from milk, meat, and vegetables. Beyond the horizon of their conventional role in acid, flavor, and texture development, they are being looked upon as burgeoning “cell factories” (Hugenholtz 2008) for production of host of functional biomolecules and food ingredients such as biothickeners, bacteriocins, vitamins, bioactive peptides, and amino acids. Besides, endowed with their relatively simple metabolism, limited biosynthetic capacity, and apparent lack of gene multiplicity, these bioreactors are ideal tool for metabolic engineering to facilitate overproduction of desirable goodies compared to other microorganisms like yeast or fungi (Kleerebezem and others 2000). To this list of metabolites, low-calorie sugars have rather been recent addition and have attracted a great deal of the interest of researchers, manufactures, and consumers for varied reasons.
Broadly speaking, low-calorie sweeteners can be categorized in to 2 groups. One group of sweeteners consists of substances with a very intense sweet taste that are used in small amounts to replace the sweetness of a much larger amount of sugar, for example, acesulfame-K, aspartame, neotame, saccharin, and sucralose, and so on. The sweeteners currently approved for use in the United States belonging to this category are acesulfame-K, aspartame, neotame, saccharin, and sucralose. Other such sweeteners used in vogue in other countries as food ingredients (not approved in the United States) are alitame, cyclamate, and stevia/steviol glycosides (Kroger and others 2006). These are produced by chemical methods and are only can be used in reconstituted foods. These sweeteners lack in bulking properties, are not globally approved and also have differing degree of after taste. Although low-calorie sweeteners are commonly associated with weight management, an emerging body of evidence on the contrary suggests that these substances offer little help to dieters and may even end up promote weight gain (Hampton 2008).
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 (http://www.22.214.171.124/news/ng.asp?id=65843-cargill-polyol-maltitol).
Polyols and other bulk sweeteners have a couple of potential advantages over sugar as food ingredients (Kroger and others 2006):
- • 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.
- • Produce a lower glycemic response than most sugars and starches do. Thus, their use may be advantageous for people with diabetes.
- • Have low calorific values due to their poor digestion and poor absorption.
In this article, an attempt has been made to discuss a number of such sugars and sugar alcohols produced by LAB in terms of their industrial application and health attributes.
Mannitol, a naturally occurring polyol (sugar alcohol with 6 carbon atoms), is found in animals and plants (Figure 1). 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 (Schwarz 1994). 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 (Soetaert and others 1999). Marine algae, especially brown algae, are also rich in mannitol (Schwarz 1994). 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 (Benson 1978). 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 (Weymarn and others 2002a). 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% (Perry and others 1997) while at 25 °C the solubility of mannitol in water is approximately 18% (Soetaert and others 1999). Mannitol is sparingly soluble in organic solvents, like ethanol and glycerol, and practically insoluble in ether, ketones, and hydrocarbons (Schwarz 1994). Mannitol forms white orthorhombic needles and the crystals have a melting point of 165 to 168 °C (Schwarz 1994). 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 (Anonymous 2002).
In our laboratory, we have initiated a study with the objectives of isolation and identification of mannitol producing Leuconostoc strains from fermented food products, optimization of cultural conditions for maximum production of mannitol, production of mannitol by fermentation and its purification, and development of a low-calorie sweetened fermented milk by coculturing Leuconostoc with Lactococcus lactis subsp. lactis.
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 (Salminen and others 1998). Mannitol has been shown to exhibit osmoprotecting effect during drying of LAB (Efiuvwevwere and others 1999). Mannitol has the potential to extend the shelf life of various foodstuffs (Soetaert and others 1999). As mannitol is only partially metabolized by humans and does not induce hyperglycemia, which makes it useful for diabetics (Griffin and Lynch 1972) and for so-called light foods. Besides, mannitol is commercially useful in making artificial resins and plasticizers.
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 (Weymarn and others 2002a). 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 (Vandamme and others 1987). Kostopanagiotou and others (2006) 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 and others (2007) 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%.
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 (Weymarn and others 2002a). 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 (Loesche and Kornman 1976), Lb. leichmanii (Chalfan and others 1975), lactate dehydrogenase-negative mutant of Lb. plantarum (Ferain and others 1996), and lactate dehydrogenase deficient mutant of Lc. lactis (Neves and others 2000).
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 (Soetaert 1990), Lactobacillus spp. B001 (Itoh and others 1992), Lactobacillus spp. KY-107 (Yun and others 1996), Lactobacillus spp. Y-107 and Leuconostoc spp. Y-002 (Yun and Kim 1998), Ln. pseudomesenteroides ATCC 12291 (Ojamo and others 2000), Lb. sanfranciscensis (Koraki and others 2000), and most recently Saha (2006b) 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 (Saha 2006a). 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 (Soetaert 1990). An improved volumetric productivity (8.9 g/L/h) but a low yield (60 mol%) was reported for cells immobilized to reticulated polyurethane foam (Soetaert and others 1990). Ojamo and others (2000) 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 and others (2002b) 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 and others (2005) 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 (Kiviharju and Nyyssola 2008).
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).
Tagatose is an isomer of fructose that occurs naturally in some dairy products (Figure 2). 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 (Levin 2002; Kim 2004). 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 (Buemann and others 1999a, 1999b, 2000; Laerke and Jensen 1999). 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 (Bertelsen and others 2001). 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 (Levin and others 1995). Although similar to sucrose in taste, it does not contribute to calorie production (Zehner and Lee 1988; Levin 2002). D-tagatose has been found not to contribute to net energy as revealed by a number of growth studies on rats (Livesey and Brown 1996; Bar and others 1999) and human clinical trials show that subjects gradually and consistently lose weight at medically desirable rates (Levin 2002). As a result of these properties, D-tagatose is considered to be a potential reduced-energy sweetener.
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 (Deok-Kun Oh 2007). Besides effecting enhancement of flavor (Rosenplenter and Mende 2004), it provides the natural taste and texture of sugar. According to Taylor and others (2008) 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 (http://www.ap-foodtechnology.com).
A number of health benefits have been attributed to tagatose such as promotion of weight loss (Buemann and others 2000), no glycemic effect (Seri and others 1995; Donner and others 1999), antiplaque, noncariogenic, antihalitosis, prebiotic, and antibiofilm properties (Cisar and others 1979; Bertelsen and others 1999; Laerke and others 2000; Wong 2000), organ transplants (Paterna and others 1998), improvement of pregnancy and fetal development (Levin 2000), treatment of obesity (Moore 2006), reduction in symptoms associated with type 2 diabetes, hyperglycemia, anemia, and hemophilia (Seri and others 1995; Levin 2002).
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 (Laerke and others 2000). 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 (Bertelsen and others 1999).
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 (Bertelsen and others 1999). 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 (Bertelsen and others 2001). 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 (Klaenhammer 1998). 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 (Gibson and Roberfroid 1995). 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 (Brand-Miller and others 2003). 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 (Frost and Dornhorst 2000; Jenkins and others 2000). Lu and others (2008) 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 and others (1998) 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 (Boelsterli 2002).
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 (Richards and Chandrasekhara 1960; Troyono and others 1992; Mendoza and others 2005). D-tagatose can be produced from D-galactose by a chemical method using a calcium catalyst (Beadle and others 1991), 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 (EC 126.96.36.199) 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 (Cheetham and Wootton 1993; Roh and others 2000). 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 (Yamanka and Wood 1966). The LAB reported to contain L-arabinose isomerase are Lb. gayonii (Nakamatu and Yamanaka 1969), Lb. plantarum, and Bifidobacterium longum (Kim 2004). Cheetham and Wootton (1993) invented a process to convert D-galactose to D-tagatose by using LAB. Further, Ibrahim and Spradlin (2000) 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 (Zhang and others 2007).
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 (Kroger and others 2006).
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 (Figure 3). Sorbitol, also referred to as D-glucitol, is naturally found in many fruits, for example, berries, cherries, and apples (Budavari and others 1996) and its worldwide production is estimated to be higher than 500000 tons/year and continues to rise (Silveira and Jonas 2002). 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.
This polyol has a relative sweetness of around 60% vis-à-vis sucrose and displays a 20-fold higher solubility in water than mannitol (Silveira and Jonas 2002). 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 (Elvers and others 1994; Silveira and Jonas 2002). Besides, sorbitol is the starting material for the production of pharmaceutical compounds such as sorbose and ascorbic acid (Budavari and others 1996). Several industrial processes have been described for the production of sorbitol (Elvers and others 1994).
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 and Kao (2003) 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.
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 (Nissen and others 2005).
Ladero and others (2007) 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.
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 (Figure 4). 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 (Crowe 2002). It acts by altering or replacing the water shell that surrounds lipid and protein macromolecules (Colaco and others 1995). 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 (Muthusamy and others 2008).
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 (Arakawa and others 2001). It is particularly effective when combined with phosphate ions (Juan and others 2003). 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.
It is anticariogenic, synthesized by several microorganisms, only partially digested by humans, and therefore considered a dietetic sugar (Ruf and others 1990; Neta and others 2000). 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.
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 (Neves 2004). Furthermore, it has been demonstrated that trehalose has important stabilizing effects on human proteins (Simola and others 2000; Benaroudj and others 2001), 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 (Colaco and others 1992). It stabilizes proteins for inhaled use (Strickley and Anderson 1997). It has been successfully used to preserve embryos (Suzuki and others 1996), and cellular blood products (Crowe and others 2003; Zhang and others 2003). Cyopreservation of transplant cells and tissue in the presence of trehalose has been shown to increase viability (Beattie and others 1997) and decrease host immune response (Erdag and others 2002).
As it inhibits lipid and protein misfolding (Singer and Lindquist 1998), 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 and others (2008) 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 and others (2003a) 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) (Kazuyuki and others 2003b).
Trehalose is widespread within the genus Propionibacterium (Cardoso and others 2004). Trehalose accumulation in Propionibacterium, for example, P. acidipropionici and P. freudenreichii subsp. shermanii (Rolin and others 1995; Pereira 1997) 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 (Cardoso and others 2007).
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 (Kroger and others 2006).
Microbial production of these sugars is becoming more attractive because of the inherent problems of the chemical processes. The examples presented here clearly demonstrate that LAB are ideal candidates for production of these low-calorie sweeteners and for metabolic engineering. A diverse number of approaches have been employed successfully to reroute carbon metabolism toward the products of interest, resulting in high-level production of both natural and novel compounds, or removal of undesirable metabolites. Some of these constructed strains will undoubtedly find their way into novel fermented products with increased nutritional value. Taking into consideration health benefits and industrial applications, the development of novel dairy products naturally enriched in these sweeteners during fermentation processes offers interesting perspectives. The polyols and similar substances used as bulk sweeteners are also safe, but consumers need to be aware of their presence in food products so that they can limit their intake sufficiently to avoid gastrointestinal discomfort. These safe sweeteners can be exploited as vital food ingredients for the development of low-calorie foods with added functional values especially for children, diabetic patients, and weight watchers.