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Journal of Food Health & Technology

Innovations June 2018, Vol.1, No.1

A New Low Calorie but it is poorly digested. It is non- Sweetener D-Tagatose cariogenic, reduces demand and from in Cheese can be used as a reduced calorie food sweetening and bulking agent. D-tagatose as a Nutraceutical is also useful as an intermediate chemical Value-Added Product for the synthesis of other optically active

Osama Ibrahim compounds and as an additive in detergent, BioInnovation LLC, Chicago, IL, USA cosmetic and pharmaceutical formulations.

Abstract In this manuscript content, a new low calorie sweetener D-tagatose from lactose The lactose in cheese in cheese whey has been described. These whey can be hydrolyzed by acid or by the research results are showing progress in improving bioconversion rate, lactase to prepare a mixture thermos ability in increasing the half-life comprising of D- and D- of the immobilized enzyme, shorting enzyme reaction time and improving both at the ratio of 1:1. D-galactose from bioconversion efficiency for the lactose hydrolysate is the substrate for the of lactose to lactose hydrolysate (D- glucose and D-galactose) and for production of D-tagatose and it can be isomerization of D-galactose to higher separated from D-glucose in lactose yield of D-tagatose. hydrolysate by column separation or by relatively converting D glucose in lactose 1. Introduction hydrolysate to ethanol by microbial An excess of lactose is currently fermentation and distillation. produced in North America dairy industry The separated D-galactose can be as a byproduct of cheese manufacture in isomerized into the low calorie sweetener the form of whey or whey permeate. These D-tagatose via chemical method or by by-products are potential source of food enzymatic method. Chemical method is for both human and animal consumption, based on using a metal hydroxide such as plus there are technologies for whey in the presence of an utilization that are being investigated. One inorganic salt catalyst to form an of these investigated methods is to convert intermediate metal hydroxide-D-tagatose the D-galactose in the disaccharide lactose complex. This intermediate complex is the in whey or whey permeate into the low then neutralized with acid to yield D- calorie sweetener D-tagatose via tagatose (Beadle et.al.,1990). Enzymatic isomerization. method is based on employing the D-tagatose is the keto of D- microbial enzyme L- isomerase galactose. It has a sweetness equivalent to

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Innovations June 2018, Vol.1, No.1 for isomerization of D-galactose into D- be used as a reduced calorie food tagatose (Ibrahim and Stradlin,2000). sweetening and baking agent. Also it is

Currently D-tagatose is generally useful as intermediate chemical for the recognized as safe by North America, synthesis of other biologically active Europe, Australian and Asia and these two chemical compounds and it can be used as manufacturing methods of D-tagatose from additive in detergents, cosmetics and the substrate D-galactose have been pharmaceutical formulations (Levin evaluated by worldwide researches for et.al.,1995). future manufacturing D-tagatose on a Functional uses of D-tagatose as large scale. food additives are as sweetener, texturizer,

Researcher’s activities are mainly stabilizer, humectant, formulation aid, and focusing on enzymatic method to improve provides several health benefits to L-arabinose isomerase properties, activity, consumers such as attenuate a glycemic and stability in order to improve response (Armstrong et.al.,2009) and manufacturing process for cost reduction. preventing tooth decay.

Reducing D-tagatose production cost is the D-tagatose received a GRAS status key factor for marketing D-tagatose as and (Generally Recognized As Safe) in United an affordable low calorie sweetener for States, Australia, New Zealand, South applications in foods, cosmetics and Korea, Brazil, South Africa, the European pharmaceuticals industries. Union and other parts of the worlds (Lina and Kuper,2002) due to its history of use

2. D-Tagatose naturally in foods with no reported adverse

D-tagatose is a keto , an effects (Adachi,1958) and for Its naturally epimer of D- (Bertelsen existing at a low concentration in human et.al.,1999) isomerized at the fourth milk, sterilized and powdered cow’s milk carbon. The chemical structure of D- (Levin et.al.,1995), fermented dairy tagatose differs from the chemical products such as yogurt (Hirst et.al.,1949) structure of D-fructose (Bueman and it is also, present as a component in et.al.,1998), only in the position of plants cell structure such as gum exudate hydroxyl group (OH) on the fourth carbon of the cacao tree (Sterculia setigera) (Elin (Figure 1). and Olarsdottir,2001) and one of

D-tagatose has approximately half component in the chemical structure of the the calorie of sucrose (1.5 kcal) and has a in lichens of the Rocella sweetness equivalent to sucrose thus it can species (Marshall and Kooi,1957)

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Innovations June 2018, Vol.1, No.1

cation exchanger. The purified D -tagatose

3. Methods of D-Tagatose fraction is crystallized (Ibrahim and

Manufacturing: Spradlin,2000), separated by

The substrate for the production of centrifugation, washed by spraying the keto sugar D-tagatose is the aldehyde distilled water and dried in drum dryer sugar D-galactose (Figure 2) (Ibrahim and Spradlin,2000; Beadle

The conversion of D-galactose into et.al,1992) D-tagatose is known by the name isomerization and this conversion process 9.3.2. Enzymatic Method can be practice by chemical isomerization Enzymes are the catalysts of using calcium hydroxide at pH12, or by biological process that bring the reaction enzymatic isomerization using the enzyme catalyzed to its equilibrium with more L-arabinose isomerase. specific reaction in short time with less

These two processes for manufacturing D- energy than the chemical method. tagatose can be summarized. Chemical method is nonspecific and require an extra steps for purification and

3.1. Chemical Isomerization decolonization to remove generated

Method impurities and color (Ibrahim and

The D-galactose at a concentration of 30% Spradlin,2000). is isomerized to D-tagatose under alkaline Converting an or aldose conditions at pH 12 for 2-4 hours using derivatives to a or ketose derivatives calcium hydroxide in the presence of an are well known. For example, the inorganic salt as catalyst to form an enzymatic conversion of D-glucose to D- intermediate “calcium hydroxide-D- fructose, using the enzyme tagatose complex”. Treatment the isomerase (EC 5.3.1.5) is widely practiced precipitated intermediate calcium on a commercial scale for the production hydroxide- D-tagatose complex with of high fructose corn syrup (Nakamatu and sulfuric acid forming calcium sulfate and Yamanaka,1969). However, the enzymatic liberates D-tagatose solution. The filtered method for converting D-galactose to D- D-tagatose solution is demineralized in a tagatose, using the enzyme L-arabinose cation and anion exchanger. The isomerase (EC 5.3.1.4) (Ibrahim and demineralized D-tagatose solution is Spradlin,2000) was not been developed concentrated and purified by until the last fifteen years as described in chromatographic fractionation using a

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Innovations June 2018, Vol.1, No.1 the granted patent for manufacturing of D- in the isomerization process depends on tagatose (Ibrahim and Spradlin,2000). the concentration of D-galactose as a

Immobilized free enzyme of L- substrate. Isomerization end products are arabinose isomerase or cell producing a mixture of D-tagatose and D-galactose. intracellular L-arabinose isomerase can be Separation of D-tagatose from D-galactose operated in a continuous, semi continuous in the mixture can be performed by or batch operation, consistent with the chromatographic fractionation using a enzyme system employed the isomerization cation exchanger column separation of 20-60 % D-galactose at PH range of methods. Separated and purified D- 5.0.5-7.0 in the presence of metallic ion as tagatose can be crystallized, centrifuged, an activator such as manganese chloride washed and dried in drum dryer. The (5mM) and optimum enzyme temperature separated D-galactose fraction from D- range of 50-80 0C (Patrick and Lee,1975). tagatose / D-galactose mixture can be The optimum pH and temperature of the recycled for enzyme isomerization to D- enzyme depends on the microbial source of tagatose. the enzyme L- arabinose isomerase. L-arabinose 3.2.1. L-arabinose Isomerase isomerase from thermophilic L-arabinose isomerase (EC 5.3.1.4) microorganisms produce thermostable is inducible enzyme (Patrick and Lee, enzyme with higher optimum temperature 1975) by the sugar L-arabinose (Sang-Jae et.al.,2005) comparing to L- and is produced as an intracellular arabinose isomerase produced from enzyme. The enzyme can be derived from mesophilic microorganisms. Thermostable various microorganisms (Sang-Jae L-arabinose isomerase has advantages et.al.,2005) include but not limited to over mesophilic L-arabinose isomerase in Lactobacillus pentosus, Lactobacillus shifting the isomerization equilibrium brevis, Lactobacillus pentoaceticus , toward the end product (D-tagatose) and Lactobacillus fermentum ( previously also, preventing the potential microbial known Lasctobacillus gayonii), Aerobacter contamination in the case of continuous aerogenes, Bacillus amyloliquefaciens, immobilization system. Bacillus subtilis, Candida utilis,

Isomerization of D-galactose into Clostredium acetobutlylicum Escherichia D-tagatose can be completed in 4-8 hours coli, Erwinniae cativosa , Pediococcus spp, depending on the enzyme activity and Arhthrobacter spp.,and Mycobacterium properties. Plus, high yield of D-tagatose smegmatis. Many of these microorganisms

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Innovations June 2018, Vol.1, No.1 are Generally Recognized As Safe (GRAS) encapsulated in its microbial, plant or and are suitable source of L-arabinose mammalian cells. There are various isomerase for the enzymatic method to immobilization techniques that are isomerize D-galactose to D-tagatose. available for L-arabinose isomerase

L-arabinose isomerase is mainly immobilization. The principle of these catalyzes the isomerization of the pentose - techniques are illustrated in (Figure 4). sugar L-arabinose to keto-sugar L- . This enzyme is also demonstrated 3.2.3. Methods of Enzymes its capability to isomerize the hexose sugar Immobilization D-galactose at high concentration into D- Cross linking: Several chemicals serve as tagatose (Manzo et.al.,,2013) (Figure 3). reagent for intermolecular are cross The high concentration of D-galactose as a linking of enzymes. Glutaraldehyde (Kim substrate for the enzyme L-arabinose et.al.,2001) .is the most popular cross- isomerase is due to the Km value for the linked reagent for both free and cellular substrate D-galactose. (Km value enzymes. expresses the binding efficiency of the Covalent binding: enzyme amino acid enzyme to its substrate). residues that are not part of the enzyme active site or substrate binding site can be 3.2.2. Enzyme Immobilization used for covalent binding with the enzyme Isomerization of D-galactose to D- support. tagatose can only be made economically Entrapment: Entrapped enzymes are feasible by immobilizing thermostable L- classified into: Lattice types (gel arabinose isomerase, at high reaction matrices), Microcapsule type (synthetic temperature to achieve a higher polymer), Liposome type (phospholipids), conversion rate of D-galactose to D- Hollow fiber type (hollow fiber), and tagatose and yield (Yan et.al.,2014) of D- Membrane type (ultrafiltration tagatose. membrane). Immobilized enzymes are defined as Encapsulation: Incorporate, enzymes or enzymes physically confined or localized in microbial cells in a micro metric scale. a certain region of space with retention of Microcapsules have pores with diameters their catalytic activities, which can be used between a few micrometers and a few repeatedly and continually. This definition millimeters: to allow the interaction is applicable to the immobilization of free between the enzyme and the substrate. enzymes (extract), or intracellular enzymes

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Adsorption::Binding enzyme to carrier by disaccharide aldehyde sugars composed of physical interaction such as hydrogen D-galactose and D-glucose with ß 1--4 bonding, hydrophobic interaction or van glyosidic bond (Figure 5).This glycoside der waal’s forces. bond that bound D-galactose to D-

galactose can be hydrolyzed to produce

lactose hydrolysate (a mixture of D-

4. Cheese Whey for the glucose and D-galactose at the ratio of Production of D-Tagatose (1:1) by acid hydrolysis method using

A total of over 90 billion pounds of strong acids such as sulfuric acid or by whey are generated in United States as a enzyme hydrolysis methos Lactase enzyme byproduct of cheese manufacturing, (E.C.3.2.1.23) is a beta-D galactoside- compromises about over 84 billion pounds galactohydrolase enzyme produced by sweet whey and a 6 billion pounds acid many microorganisms. Lactase enzyme is whey at the ratio of 9:1 (Charles intracellular enzyme and can be extracted

Ling,2008). The average composition of commercially from yeasts such as whey is approximately 0.3 % butterfat, 0.8 Kluyveromyces fragilis and

% whey protein, 4.9 % lactose and 0.5% Kluyveromyces lactis or from molds, such minerals (USA Wisconsin Center for Dairy as Aspergillus niger and Aspergillus Research). Cumulatively, there are over oryzae. The commercial enzyme lactase 4.1 billion pounds of lactose in cheese has an optimum temperature of over 48 °C whey that can be utilized for the and an optimum pH of 6.5. production of D-tagatose. Currently, there The hydrolysis of lactose can be are about 726 million pounds of lactose summarized as follow; 20-40 % lactose per year are recovered in United States powder in water is subject for acid or from whey. enzymatic hydrolysis. Acid hydrolysis

Whey is the major byproduct from method (Douglas et.al.,2001) can be cheese processing industry and it is carried out using 0.1% w/w of sulfuric acid estimated that for the production of one and incubation at 1000C for 1-2 hours pound of cheese it will generate a nine followed by neutralization to PH 5.5-7.0. . pounds of whey. The current lactose Enzymatic hydrolysis method (Demaimay crystal/ powder price is in United States is et.al.,1978) can be carried out using in the range of 20 cents per pound. The Lactase enzyme by conventional means chemical structure of lactose (β-D- (free enzyme in batch process) or a galactopyranosyl-(1→4)-D-glucose) is a continuous (immobilization process), at

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Innovations June 2018, Vol.1, No.1 optimum temperature 40-600C and depends on the initial concentration of the optimum PH 4-6 (the enzyme optimum substrate D-galactose.

conditions depends on the microbial source and the properties of lactase enzymebeing used). 5. D-Tagatose Properties

The end product after lactose D-tagatose is virtually odorless, hydrolysis by either method (acid or white or almost white, non-hygroscopic enzyme) is lactose hydrolysate which is crystals and it has almost the same compromise of a mixture of D-galactose sweetness as sucrose with less than half the and D- glucose at an equal quantities calories of sucrose (Figure 8). It is (Figure 6). naturally exist as a keto

For the production of D-tagatose, sugar with unique properties. The standard the substrate D-galactose can be separated specification of D-tagatose is illustrated in from D-glucose by a column separation (Table 1). using a cation exchange resin or by D-tagatose provide health benefits selective fermentation of the D- glucose to to consumers such as having prebiotics ethanol using bacteria or yeast strains function, attenuate glycemic response (Ibrahim and Spradlin,2000). In the case (Donner et.al.,1999), does not promote of D-glucose fermentation to ethanol, tooth decay (Levin, 2002; Livesey and microorganisms for ethanol production Brown,1996) and has poor absorption in consumes first the sugar D-glucose for GI and low metabolizing energy. These the production of ethanol and the properties of D-tagatose gained fermentation should terminated as soon the international attention as a low calorie sugar D- glucose is completely consumes sweetener for diabets, obese and for weight into ethanol. The produced ethanol from consciousness population. sugar D-glucose can be separated the D-tagatose is stable in food remaining D-galactose by distillation and products during processing and storage. It the remained D-galactose is concentrated is stable at both room and cold to 40-70 % to be used as a substrate for temperature. D-tagatose as the isomerization process into D-tagatose undergo Maillard browning reaction with by chemical or enzymatic method (Figure amino acids that gives brown color to 7). The D-tagatose production yield is vary baked good (FAO 2004) from 20-30%

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6. D-Tagatose Metabolism This enzyme fructokinase is the same

Only about 20% of tagatose is enzyme that phosphorylate fructose in absorbed in the small intestine, the rest fructose metabolism into fructose-1- passes to the where it is phosphate. fermented by the beneficial microflora in Similar to fructose -1- phosphate, the colon into short chain fatty acids such the tagatose -1-phosphate is an inhibitor of as butyric acid which helps in preventing the enzyme phosphorylase from colon cancer (Johansen and and causing glycogen to remain stored in the Jensen,1997). These short chain fatty acid liver as a mechanism of action for glycemic are absorbed into blood stream from the control. However, the cleavage of tagatose Intestinal tract (Bruemann et.al.,1998), as -1-phosphate is slower than fructose -1- a source of energy. Also, D-tagatose phosphate, therefore D-tagatose ingestion blocks the digestion of sucrose, , may cause a longer lasting reduction in and other causing a small inorganic phosphate (Pi) and adenosine or no increase of glucose level in blood triphosphate (ATP) levels in the liver stream after meals (Lu et.al.,2008). comparing to D-fructose metabolism

The metabolism of D-tagatose, (Bar,2000). which is a stereoisomer of D-fructose is in Tagatose -1-phoshatein in the the liver as fructose metabolism (Buemann tagatose pathway is then undergoes et.al.,2000). It convert into glucose which hydrolysis by the enzyme tagatose-1 stored in the form of glycogen or broken phosphate aldolase to form down to produce energy. In addition D- phosphate (DHAP) and tagatose promote the metabolism of . The formed

Glucose-6- Phosphate which stimulates dihydroxyacetone phosphate (DHAP) is and maintain the storage of glucose in the isomerized to glyceraldehyde 3-phosphate form of glycogens (Espinosa and by the enzyme phosphate isomerase Fogelfeld,2010) stored in the liver. and the formed glyceraldehyde is

It is hypothesized that D-tagatose is phosphorylated to glyceraldehyde 3- metabolized in the liver like fructose with phosphate by the enzyme glyceraldehyde similar pathway, but at a lower rate. .The kinase. The two similar metabolites of first step in the metabolism of D-tagatose glyceraldehyde -3- phosphate from one in the liver as illustrated in (Figure 9) is molecule of D-tagatose are the the phosphorylation of tagatose to tagatose intermediate in glycolysis pathway that -1- phosphate by the enzyme fructokinase. leads to:

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1 Glycogen synthesis, as nausea, flatulence and diarrhea. In

2 Oxidized to pyruvate pathway. addition, since D-tagatose is metabolized

3 Reduced to lactate. the same way as D-fructose, it is probably

4 Acetyl CoA for the anaerobic citric not safe to use by individuals with acid cycle in the mitochondria, for hereditary fructose intolerance (HFI). energy generation in the form of However accidental intake of small amount ATP. of D-tagatose is not likely harmful. In addition, D-tagatose does not likely trigger

7. D-Tagatose Applications allergic reaction in individuals with milk

D-tagatose has a potential multiple allergy (lactose intolerance) and in applications in foods such as cereals, diet compare to D-fructose it is slightly soft drinks, confectionary, hard and soft increases uric acid levels, but there is no confectionaries, backed goods, ice cream, evidence that it could increases the risk of chewing gum, ready-to-eat breakfast, etc., gout (Bar,2004). as illustrated in (Table 2). In addition to the above foods use. D-tagatose has the 9. D-Tagatose and Health potentially for use as a low calorie oral Benefits prescription drugs additive to mask Consumption of D-tagatose provides unpleasant taste. the following multiple health benefits along with antioxidant property, the treatment of

8. D-Tagatose Safety and control obesity:

D-tagatose is Generally Recognized 1. Antidiabetic potential: promoting As Safe (GRAS) by the US Food and Drug glycogen synthesis and decrease Administration (FDA) (Bar,2000). Plus, the glycogen utilization. Also , attenuate World Health Organization’s Joint Expert intestinal glucose absorption

Committee on Food Additives (JECFA) has 2. Weight loss: has net zero value left Acceptable Daily Intake (ADI) for 3. Increase in HDL: helps rise the tagatose “unspecified,” which means that good cholesterol (HDL) and may high intake of D-tagatose does not have any prevent heart attack. expected long-term toxic effects (FDA,2003). 4. Prebiotic effect: only 20 % of ingested D-tagatose absorbed into

In sensitive individual, D-tagatose bloodstream while the remaining when is taken in doses over 10-15 gm / day 80% promote the growth of could cause mild digestion problems such beneficial bacteria in the colon.

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5. Dental Health: non-fermentable and whey to keto sugar D-tagatose have not it does not promote tooth decay. been developed on a large bench scale

until recently (Ibrahim and

10. Conclusions Spradlin,2000)..

D tagatose is a rare natural An estimated of over 90 billion monosaccharide which can be pounds of cheese whey are generated in manufactured by the chemical or United States as a byproduct of cheese enzymatic isomerization of D-galactose. D- production and relatively small volume of galactose can be obtained by acid or cheese whey are converted into products enzymatic hydrolysis of milk disaccharide with various processes and technologies sugar lactose, into lactose hydrolysate (D- such as condensed whey, dry whey, dried galactose and D-glucose at ratio 1:1), The modified whey, whey protein concentrate , D-galactose is the substrate for D-tagatose whey protein isolate, lactose permeate and manufacturing and can be separated from crystalized or dried lactose. The rest of D-glucose by column separation or by unprocessed cheese whey are used for selectively converting D-glucose to animal feed rand for land spreading. ethanol. Ethanol can be removed via Crystalized or powder lactose is the distillation as a valuable byproduct. The best pure feedstock for the two steps separated aldehyde hexose sugar D- processes in D-tagatose manufacturing galactose can be isomerized to keto hexose from whey. These two steps for D-tagatose sugar D-tagatose by chemical or enzymatic manufacturing from whey are lactose isomerization. . Enzymatic isomerization of hydrolysis and D-galactose isomerization. D-galactose to D-tagatose is by the enzyme In addition to pure lactose crystal or

L-arabinose. Isomerase. Enzyme powder as a feedstock for D-tagatose isomerization is the most promising future manufacturing ,the concentrated lactose process for the large scale production of permeate after the removal of whey protein D-tagatose. and other solids can be used as a feedstock

Enzymatic methods for the in these two steps process for D-tagatose isomerization of aldose to ketose are manufacturing . Concentrated lactose widely practiced on a commercial scale for permeate consists mainly of 65-85 % converting aldehyde sugar D-glucose from lactose and salts. Utilization of lactose to keto sugar D- fructose, however permeate is the cheapest feedstock than this enzymatic method for converting lactose crystal or powder for D-tagatose aldehyde sugar D-galactose from cheese production but required an extra step in

20 Journal of Food Health & Technology

Innovations June 2018, Vol.1, No.1 the process for salts removal by risks associated with obesity, and desalination process (Ibrahim and reduction of dental caries.

Spradlin,2000) using reverse osmosis or Reported health benefits associated ultrafiltration technology. Concentrated with of low calorie sweeteners resulted in lactose permeate contain over 7% ashes. the increasing worldwide demands for such

D-tagatose is listed as a GRAS sweeteners in foods and beverages, and the ingredient in United States., Europe, 2016 global market analysis for low Australia and Asia, and in the year 2003 calorie food products and beverages is the United States Food and Drug reaching to over $ 9.0 billion and the Administration (U.S .FDA) has allowed a majority of these products containing high health claim for D-tagatose and the risk intense zero calorie artificial sweeteners reduction of dental caries. In addition, the such as aspartame, and , or low European Union (EU) has been formally calorie sugar alcohols such as sorbitol and approved D-tagatose as a “novel food xylitol. Up- to- date the natural low calorie ingredient) without any restriction on sweetener D-tagatose is failed to compete usages. Plus the joint FAO /WHO Expert in this market share of low calorie Committee on Food Additives (JECFA) products due to its production cost that concluded that D-tagatose was-not estimated to be in the range of $4-6 per genotoxic, embryo-toxic or teratogenic, pound and the estimated market price is and identified a NOEL of 075 gm / kg body over $10 per pound. This high market price weight (45 gm. D-tagatose/ 60 kg body of D-tagatose making difficulties for the weight) for human intake. application of D-tagatose in low calories

The D-tagatose safety is also due to food products and beverage its long history of safe use as one of the There is a potential in reducing the components in natural foods that production cost of D-tagatose as a result of demonstrated no indications of any the ongoing research in fields of molecular significant adverse effects related to D- biology, enzyme engineering and tagatose was published in the publically bioprocessing. These ongoing research is available literature. aimed to improve the two enzymes lactase

Health professional and consumers (B-D- galactosidase) and L-arabinose believe that low calorie sweeteners isomerase expression (Bueman including D-tagatose are effective for et.al.,1998), activities, specificities (Marta weight maintenance, weight reduction, and Jozef,2012) and thermostability (Yan managements of diabetes, reduction of et.al.,2014) for the two steps process for

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Innovations June 2018, Vol.1, No.1 the production of D-tagatose from lactose expenditure or respiratory exchange ratio. in whey. J Nutr 128:1481–1486.

These research results are showing Levin, G.V., Zehner, L.R., Saunders, J.P. & progress in improving enzymes Beadle, J.R.1995. Sugar substitutes: their bioconversion rate, thermos ability in energy values, bulk characteristics, and potential health benefits. Am. J. Clin. increasing the half-life of the immobilized Nutr., 62(5 Supple): 1161S-1168S. enzyme, shorting enzyme reaction time and

Armstrong, M. Laura, Katherine J. Luecke improving both bioconversion efficiency & Leonard N. Bell. 2009. Consumer for the hydrolysis of lactose to lactose evaluation of bakery product flavour as affected by incorporating the prebiotic hydrolysate (D-glucose and D-galactose) tagatose. International Journal of Food and for isomerization of D-galactose to Science and Technology, 44, 815–819. higher yield of D-tagatose. These Lina B.A.R. and Kuper C.F. 2002. Chronic researches progress will reduce the D- toxicity and carcinogenicity study with D- tagatose. TNO Nutrition and Food tagatose production cost (Kim et.al.,2001) Research, The Netherlands, Report Number and might bring hope for the future V 4533. marketing this natural low calorie Adachi S.1958. Formation of and sweetener (D-tagatose) with health tagatose from lactose in strongly heated benefits into the market and potential milk. Nature 181:840–841. applications in flow calories products Levin, G.V., Zehner, L.R., Saunders, J.P. & (Marta and Jozef,2012). Beadle, J.R. 1995. Sugar substitutes: their

energy values, bulk characteristics, and References potential health benefits. Am. J. Clin. Nutr., 62 (5 Supple): 1161S-1168S. Beadle JR, Saunder JP, Wajada TJ. 1992. Process for manufacturing tagatose. US Hirst E.L., Hough L., and Jones J.K. N. Patent 5,078,796. 1949.The structure of Sterculla Setigera

gum. Part 1: The investigation by the Ibrahim OO, Spradlin JE. 2000. Process for manufacturing D-tagatose. US Patent method of paper partition chromatography 6,057,135. of the products of hydrolysis of the gum. J. chem. Soc. 3145-3151. Bertelsen, H., Jensen, B.B. & Buemann, B.1999. D-Tagatose –a novel low-calorie Elin S., Olarsdottir Kristine Igolfsdottir. bulk sweetener with prebiotic properties. 2001. from Lichens: World Rev. Nutr. Diet, 85: 98-109. structural, characteristics, and biological

Buemann B, Toubro S, Astrup A. 1998. D- activities. Planta Med. 67: 199-208. Tagatose, a stereoisomer of D-fructose,

increases hydrogen production in humans without affecting 24-hour energy

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Marshall R., Kooi E.1957. "Enzymatic Business and Cooperative, program, conversion of D-glucose to D-fructose". Research report 214 February 2008.

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Demaimay M., LeHenaff Y, and Printemps Patrick, J. & Lee, N. 1975. L-arabinose isomerase. Methods in Enzymology 41, P. 1978. Hydrolysis of Lactose on catalytic Resin. Process Biochemistry 3-5. 453-458 Donner T.W, Wilber J.F., and Ostrowski D. Sang-Jae Lee1,Dong-Woo Lee ,Eun-Ah 1999. D-tagatose a novel hexose: Acute Choe, Young-Ho Hong,Seong-Bo Kim, effects on tolerance in subjects with and without type 2 diabetes. ,Byoung-Chan Kim and Yu-Ryang Pyun. 2005. Characterization of a Diabetes, Obesity and metabolism 1:285- 29. Thermoacidophilic L-Arabinose Isomerase from Alicyclobacillus acidocaldarius: Levin G. V. 2002. Tagatose, the new GRAS Appli. Environ. Microbiol. 71 (12), 7888– sweetener and health product. J. Med 7896. Food, 5,23-36.

Manzo R.M., Simonetta A. C., Rubiolo A. Livesey G., Brown J.C. 1996. D-tagatose is a bulk sweetener with zero energy C., Mammarella E. J. 2013. Screening and determined in rats. J. Nutr.126, 1601-1609. selection of wild strains for L-arabinose isomerase production. Braz. J. Chem. Eng Food and Drug Administration (FDA). . Vol. 30, No. 04, 711 – 720. 2004. D-tagatose. Yan Men, Yueming Zhu, Lili Zhang, , Zhenkui Kang, Ken Izumori,, Yuanxia Sun,, Johansen H.N., and Jensen B.B. 1997. and Yanhe Ma. 2014. Enzymatic Recovery of energy as SCFA after conversion of D-galactose to D-tagatose: microbial fermentation of D-tagatose. Int. Cloning, overexpression and J. Obes. (suppl.2) 21:S50 (abs). characterization of L-arabinose isomerase from Pediococcus pentosaceus PC-5 . Buemann B, Toubro S, and Astrup A. 1998. Microbial Research. 169, Issues 2–3, 171- Tagatose a stereoisomer of D-tagatose a 178. stereoisomer of D-fructose, increases

Kim P, Yoon SH, Roh HJ, Choi JH. 2001. hydrogen production in human without affecting 24-hour energy expenditure of High production of D-tagatose, a potential , using respiratory exchange ratio. J. Nutr. immobilized L-arabinose isomerase. 128:1481-1486. Biotechnol. Prog .17: 208-210 (2001) Lu Y. G. V Levin, and T.W. Donner. 2008. Charles Ling K.2008. Whey to ethanol: A Tagatose, a new antidiabetic and obesity biofuel role for cooperative? .Rural

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Innovations June 2018, Vol.1, No.1 control Drug, diabetes, Obesity, and conversion of D-galactose to D-tagatose: Metabolism 10: 109-134. Cloning, overexpression and characterization of L-arabinose isomerase Buemann B, Gesmar H., Astrup A, and from Pediococcus pentosaceus PC-5 . Quistorff B. 2000. Effects of oral D- Microbial Research. 169, 2–3, 171-178. tagatose, a stereoisomer of D-fructose, on liver metabolism in man as examined by 3 Kim P, Yoon SH, Roh HJ, Choi JH. 2001. PMRS. Metabolism 49:1335-1339. High production of D-tagatose, a potential sugar substitute, using Espinosa I., and Fogelfeld L. 2010. immobilized L-arabinose isomerase. Tagatose; from sweetener to a new Biotechnol. 17, 208-210. diabetic medication? Expert Opin Investig Drugs 19: 285-294.

Bar A. 2000. Dossier prepared on behalf of Arla Food ingredients amba, Viby, Denmark for safety assessment of D- tagatose by a panel of independent experts. Cited in GRN 000078.

Food and Drug Administration (FDA). 2003. Food labeling. Health claims. D- tagatose and dental caries. Final rule. Fed Reg. .68:39831-39833.

Bar A. 2004. Tagatose: Food Scientific Regulatory Service. Bioresco.

Buemann B., Toubro S., Holst J. J., Rehfeld J., and Astrup A. 1999. D- tagatose stereoisomer of D-fructose , increases blood uric acid concentration . Metabolism 49: 969-976.

Marta W., and Jozef K. 2012. A method for the production of D-tagatose a recombinant Pichia pastoris strain

secreting B-DD-galactosidase from Arthrobacter chlorophenolicus and a recombinant L-arabinose isomerase from Arthrobacter sp.22cMicrob.Cell Fact. 11- 13.

Yan Men, Yueming Zhu, Lili Zhang, , Zhenkui Kang, Ken Izumori,, Yuanxia Sun,, and Yanhe Ma. 2014. Enzymatic

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Innovations June 2018, Vol.1, No.1

A new low calorie sweetener [Galactose is a monosaccharide sugar that D-tagatose from lactose in is less sweet than glucose and fructose. It is a C-4 epimer of glucose]

cheese whey as a nutraceutical

value-added product

FIGURES

Figure (3): L-arabinose isomerase

mechanism of action

[Isomerization of L-arabinose to L- ribulose and D-galactose to D-tagatose]

Figure (1): D-tagatose vs, D-fructose

[D-tagatose differs from D-fructose only in th the position of hydroxyl group (OH) on 4 carbon]

Figure (4): Enzymes Immobilization methods

Figure (2): D-galactose chemical [Cross linking, covalent binding, structure entrapment encapsulation and adsorption],

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June 2018, Vol.1, No.1

Figure (5): Milk sugar lactose

[Lactose; β-D-galactopyranosyl-(1→4)-D- glucose is a disaccharide sugar composed of galactose and glucose that is found in milk]

Figure (6): Acid or enzymatic hydrolysis of lactose

[Lactose hydrolysis to D-galactose and D- glucose at ratio 1:1]

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Innovations June 2018, Vol.1, No.1

Chemical method TABLES Enzymatic method

Figure (7): D-tagatose manufacturing process Parameter Specifications [Diagrams for D-tagatose manufacturing from lactose in whey by two methods] D-tagatose- 98.5% (wt. /wt.) \ D-galactose & other sugars

≤ 1% (wt. /wt.)

Moisture

- ≤ 0.5% (wt. /wt.)

Ash ≤ 0.1 % (wt. /wt.)

Solubility in water: at 20 0 Figure (8): D-tagatose sweetness and C 160 mg / 100 ml. calorie comparing to others sugars Solubility in ethanol: a t 22°C [D-tagatose is sweet as sucrose and has 0.02 gm / 100 ml

1.5 kcal] Melting point range 133 – 137 °C

Total plate count

<10,000 CFU / g

Coliform

Negative

Staphylococcus aureus Negative

Heavy metals (Hg, Cd, As)

<0.1 ppm

Figure (9): D-tagatose metabolism Lead < 0.5 ppm [D-tagatose metabolism has a similar pathway as fructose] Physical appearance White Crystal

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Innovations June 2018, Vol.1, No.1

Table 1: D-tagatose finish product Biscuits 10. [Standard specifications] Cookies 10.

Cereal bar

. 10.

Food Category Maximum level of use (%) Table 2: The proposed food applications of D- tagatose and maximum level of use Ready-to eat Breakfast cereal

33 [WHO’s Joint Expert Committee on Food Additives Diet soft drinks (JECFA) has left Acceptable Daily Intake (ADI) for 2 D-tagatose “unspecified,” which means that high intake of D-tagatose does not have any expected long- Non-diet soft drinks 3. term toxic effects]

Confectionary 25

Formulated diets for meal

replacement 3.

Meal replacement drink mix (powder)

33

Cake, Pie 10.

Cake mixed powder 15.

Frostings

15.

Ice cream and frozen yogurt

7.5

Yogurt 7.5

Chewing gum, sugar free 60.

Jelly and pudding

7.5

Coffee mix powder

7.5

28