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Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Progress in the Understanding of Transgalactosyl Reactions
  5. Substrate Sources for GOS Synthesis
  6. GOS Yields Using Different Enzyme Sources
  7. Glycosyl-Linkage Analysis of GOSs
  8. Determinants of GOS Composition, DP and Glycosyl Linkages during Synthesis
  9. The Effect of Fermentation Parameters on the Yield of GOS Synthesis
  10. Role of Thermostable Enzymes in GOS Synthesis
  11. Analysis of GOSs
  12. Possible Ways of Improving GOS Yield
  13. Safety Aspects of GOSs
  14. Applications of GOSs as Functional Food Ingredients
  15. Future Prospects
  16. Acknowledgments
  17. References

Abstract:  Galactooligosaccharides (GOSs) are nondigestible oligosaccharides and are comprised of 2 to 20 molecules of galactose and 1 molecule of glucose. They are recognized as important prebiotics for their stimulation of the proliferation of intestinal lactic acid bacteria and bifidobacteria. Therefore, they beneficially affect the host by selectively stimulating the growth and/or activity of a limited number of gastrointestinal microbes (probiotics) that confer health benefits. Prebiotics and probiotics have only recently been recognized as contributors to human health. A GOS can be produced by a series of enzymatic reactions catalyzed by β-galactosidase, where the glycosyl group of one or more D-galactosyl units is transferred onto the D-galactose moiety of lactose, in a process known as transgalactosylation. Microbes can be used as a source for the β-galactosidase enzyme or as agents to produce GOS molecules. Commercial β-galactosidase enzymes also do have a great potential for their use in GOS synthesis. These transgalactosyl reactions, which could find useful application in the dairy as well as the larger food industry, have not been fully exploited. A better understanding of the enzyme reaction as well as improved analytical techniques for GOS measurements are important in achieving this worthwhile objective.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Progress in the Understanding of Transgalactosyl Reactions
  5. Substrate Sources for GOS Synthesis
  6. GOS Yields Using Different Enzyme Sources
  7. Glycosyl-Linkage Analysis of GOSs
  8. Determinants of GOS Composition, DP and Glycosyl Linkages during Synthesis
  9. The Effect of Fermentation Parameters on the Yield of GOS Synthesis
  10. Role of Thermostable Enzymes in GOS Synthesis
  11. Analysis of GOSs
  12. Possible Ways of Improving GOS Yield
  13. Safety Aspects of GOSs
  14. Applications of GOSs as Functional Food Ingredients
  15. Future Prospects
  16. Acknowledgments
  17. References

Galactooligosaccharides (GOSs) are regarded as an emerging special class of prebiotics that is primarily synthesized from lactose. It is the main milk sugar, and is associated with dairy products but could also be made into pure solutions. It is the main substrate for GOS synthesis, through a reaction process referred to as transgalactosylation. Transgalactosylation is the process by which the enzyme β-galactosidase hydrolyzes lactose and, instead of transferring the galactose unit to the hydroxyl group of water, the enzyme transfers galactose to another carbohydrate, in this case lactose, to result in oligosaccharides with a higher degree of polymerization (DP) (Kim and others 1997). These molecules formed by galactose transfer contain one or more galactosides that are named (GOSs) (Petzelbauer and others 2000a). Since transgalactosylation products are substrates of β-glycosidase-catalyzed hydrolysis, the composition of the product mixture changes quite significantly with progressing reaction time, hence the significance of an optimized reaction process to monitor GOS yield with reaction time (Boon and others 1999). The other reaction that lactose easily undergoes is hydrolysis in which the molecule is split into the monomeric forms of glucose and galactose. Lactose hydrolysis and transgalactosylation are concomitant reactions catalyzed by β-glycosidase, resulting in monomeric products, as well as many newly formed β-glycosides, mainly di-, tri-, and tetrasaccharides (Prenosil and others 1987).

Prebiotics are nondigestible food ingredients that reach the colon thus stimulating the growth or activity of bacteria in the digestive system that are beneficial to the health of the body (Gibson and Roberfroid 1995). Extensive studies have revealed that oligosaccharides, which may reach the lower digestive tract without being absorbed, can be utilized by bifidobacteria as an energy source and promote the proliferation of intestinal bifidobacteria (Onishi and Tanaka 1995). Equally, GOS can also serve as an important growth factor in the proliferation of other probiotic intestinal bacteria such as Lactobacillus acidophilus and L. casei. These oligosaccharides are also collectively referred to as bifidus factors or prebiotics. They have been known to promote and sustain the growth of beneficial bacteria, especially bifidobacteria within the colon (Tomomatsu 1994). The observation that GOS can stimulate the growth of bifidobacteria and other health-promoting bacteria (Rabiu and others 2001) has generated interest in the transferase reaction of β-galactosidases (Cho and others 2003). Also, it has been reported that GOS in a commercial milk powder can support the in vitro growth of 2 strains of probiotic bacteria, Bifidobacterium lactis DR10 and L. rhamnosus DR20. These studies therefore confirm the positive effects of GOS on digestive health upon consumption. Among the breast-fed infants, where the intestinal microflora is not yet well developed, their improved colonic health has been attributed to GOS in their mothers’ milk (Boehm and others 2004). Among older consumers, other health benefits linked to GOS consumption include reduction in colon cancer risk and enhanced immunity (Crittenden and Playne 1996). Short-chain fatty acids (SCFAs) are key products of GOS fermentation in the colon and the profiles of propionate and butyrate concentrations vary from one oligosaccharide to another. Propionate and butyrate have been confirmed as having positive implications in the prevention of colon cancer (Cummings 1981). Currently, there is much interest in the concept of active management of colonic microflora with the aim of improving human health. This has been attempted by the consumption of live microbial food components or supplements, known as probiotics. An alternative approach, however, is the consumption of prebiotics that provide nourishment to intestinal bacteria thus promoting their proliferation.

β-Galactosidase, also known as lactase, is a hydrolase that attacks the O-glucosyl group of lactose. The enzyme is derived from various microorganisms, including fungi and bacteria, and has been found to perform different degrees of transgalactosyl bioconversions leading to variations in the level and composition of GOSs synthesized. Probiotic microorganisms might therefore be used to produce GOS structures that could have special prebiotic effects, specifically targeting colonic probiotic strains (Rastall and Maitin 2002) and consequently improve the host's immunity. Ideally, the use of probiotic microorganisms with high β-galactosidase activity could be very important in the development of functional compounds such as GOS. According to the current definition adopted by the Food and Agriculture Organization (FAO) and World Health Organization (WHO), probiotics are: “Live microorganisms which when administered in adequate amounts confer a health benefit on the host” (Joint FAO/WHO Report 2009). Lactic acid bacteria (LAB) and bifidobacteria are the most common types of microbes used as probiotics; but certain yeasts and bacilli may also be important. Probiotics are therefore commonly consumed as part of fermented foods with specially added active live cultures, such as in yogurt, soy yogurt, or as dietary supplements. In the case of use as enzyme sources for GOS synthesis, they could provide the double advantage as probiotics as well as in prebiotic synthesis.

Prenosil and others (1987) showed that of all β-galactosidases from fungal sources, that from Aspergillus oryzae produced the highest concentration of GOS compared with that from A. niger, Kluyveromyces lactis, and K. fragilis. The yeast Sterigmatomyces elviae CBS8119 has been found to be the highest producer of GOS among yeasts (Onishi and Tanaka 1995). Earlier, Nakanishi and others (1983) had shown that β-galactosidase from Bacillus circulans was the best for GOS production among bacterial sources when compared with that from Escherichia coli and yeasts.

Chemical synthesis of GOS is also possible but it requires many reaction steps due to the necessary selective protection of the hydroxyl groups, which is not the case with enzymatic synthesis. Also, the environmental impact of toxic reagents would be far greater with chemical than with enzymatic synthesis (Hansson and others 2001). Therefore, enzymatic processes are more feasible, more environmentally useful, and less costly than the chemical processes. Consequently, there remains an untapped potential as yet for GOS (bio)synthesis (Gekas and Lopez-Leiva 1985) from pure lactose solutions and low-value whey lactose from the dairy industry to create high-value functional food products. Apart from GOS, the other carbohydrates reported to have prebiotic properties are the isomaltooligosaccharides (IMO), fructooligosaccharides (FOS), lactulose (Gibson and others 1999), and xylooligosaccharides (Suwa and others 1999).

In this review, the biosynthesis of the prebiotic GOS using β-galactosidase of bacterial, fungal, and yeast origins is extensively examined and a concise summary is provided to stimulate further interest in research and industrial applications in this area.

Progress in the Understanding of Transgalactosyl Reactions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Progress in the Understanding of Transgalactosyl Reactions
  5. Substrate Sources for GOS Synthesis
  6. GOS Yields Using Different Enzyme Sources
  7. Glycosyl-Linkage Analysis of GOSs
  8. Determinants of GOS Composition, DP and Glycosyl Linkages during Synthesis
  9. The Effect of Fermentation Parameters on the Yield of GOS Synthesis
  10. Role of Thermostable Enzymes in GOS Synthesis
  11. Analysis of GOSs
  12. Possible Ways of Improving GOS Yield
  13. Safety Aspects of GOSs
  14. Applications of GOSs as Functional Food Ingredients
  15. Future Prospects
  16. Acknowledgments
  17. References

Lactose hydrolysis and transgalactosylation are complex processes involving a multitude of sequential reactions with saccharides as intermediate products as well as the formation of GOS apart from glucose and galactose as shown in Figure 1. As can be seen, the normal function of β-glycosidases is to hydrolyze substrates formed by a monosaccharide coupled by a β bond (β1′4 and β1′6 more common and β1′2 and β1′3 rarely) to another polyol (Figure 1). However, under certain conditions, the same enzymes also catalyze the transgalactosylation reaction and synthesize GOS. The main drawback of oligosaccharide synthesis by these enzymes is that the reaction equilibrium is shifted to favor hydrolysis over synthesis in aqueous systems, which leads to a low yield in GOS production (Petzelbauer and others 2000b).

image

Figure 1–. Transgalactosylation and hydrolytic processes involving lactose and β-galactosidase to produce forms of galactooligosaccharides.

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Hydrolysis and possibly the transgalactosyl reaction of lactose by E. coli β-galactosidase was first postulated by Wallenfells and Malhotra (1960). They first suggested that glucose liberation was due to the hydrolysis of the D-pyranoside ring as opposed to the glycosidic bond. The untouched glycoside bond could, however, cause inhibition by blocking the active site of the enzyme. The authors were only able to analyze monomeric sugars; however, they eventually quantified sugar oligomers beginning with allolactose as a primary intermediate product. Allolactose, being similar to lactose, except having a β1′6 instead of a β1′4 glycoside bond, led to the conclusion that the enzyme had an extra role in bond modification. Further investigation of the transgalactosyl reaction led Jobe and Bourgeois (1972) to conclude that β-galactosidase could have different binding sites for the 1′4 and 1′6 lactose isomers. Other investigators, such as Nishizawa (1960) and Nisizawa and Hashimoto (1970), postulated that hydrolytic potential of lactose depended on the isomeric orientation of lactose and that it increased in the order of 1′3, 1′4, and 1′6. Further investigation on the kinetics of the lactose reaction included studies on the specific rotation of galactose (Huber and others 1981). But it was Flaschel and others (1982) who analyzed the specific rotation of both α- and β-glucopyranose in lactose as it approached the equilibrium between its α- and β-anomeric forms, and they concluded that β-galactosidase from E. coli hydrolyzed α-lactose 2 times faster than the β form. Escherichia coli, whose plasmids can also be used to up-regulate the production of β-forms of GOS from lactose (Ji and others 2005) apparently possess higher α-lactase activity than β-lactase activity. Between lactose and galactose, the latter was found to be a stronger inhibitor than the former for the active sites of β-galactosidase by Flaschel and others (1982), who also found that α-galactose was a more significantly stronger inhibitor than the β-form.

According to Prenosil and others (1987), β-galactosidase action on lactose and the products of its hydrolysis brought much insight into the reaction mechanism, but, on the other hand, made the classical kinetic modeling approach impractical due to the complexity of the equations and the number of the kinetic constants involved. Until the recent past, the reaction mechanism for GOS synthesis had not been fully understood (Vasella and others 2002). Reactions involving lactose and β-galactosidase are initiated by a covalent bonding of the galactosyl moiety to the active site of the enzyme, thus releasing the glucose moiety. Two types of reactions can possibly occur, namely, hydrolysis and transgalactosylation. In the case of GOS synthesis, transgalactosyl reaction is desired as opposed to hydrolysis. The determinant of either reaction is the galactosyl acceptor in the reaction system. When the acceptor is a sugar, then there is a galactosyl transfer favoring GOS formation. Conversely, when the acceptor is water, then hydrolysis occurs and lactose is broken down into its constituent monomeric glucose and galactose. The transgalactosyl reaction model can be applicable in the dairy industry where milk sugar can be used as suitable substrate for GOS synthesis, thus expanding the niche of valuable products obtained from milk. A recent study by Curda and others (2006) established that GOS can be obtained and purified from dried buttermilk using a purified β-galactosidase. Varying concentrations of the enzyme (Maxilact LX 5000) ranging from 0.1% to 2% (vv−1) can be used to obtain a maximum of 31.4% GOS (ww−1).

Substrate Sources for GOS Synthesis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Progress in the Understanding of Transgalactosyl Reactions
  5. Substrate Sources for GOS Synthesis
  6. GOS Yields Using Different Enzyme Sources
  7. Glycosyl-Linkage Analysis of GOSs
  8. Determinants of GOS Composition, DP and Glycosyl Linkages during Synthesis
  9. The Effect of Fermentation Parameters on the Yield of GOS Synthesis
  10. Role of Thermostable Enzymes in GOS Synthesis
  11. Analysis of GOSs
  12. Possible Ways of Improving GOS Yield
  13. Safety Aspects of GOSs
  14. Applications of GOSs as Functional Food Ingredients
  15. Future Prospects
  16. Acknowledgments
  17. References

Lactose accumulates worldwide at an estimated 3.3 million metric tons annually, as a component of dairy byproducts, especially from cheese whey; and approximately half of that is used for human consumption or animal feed, and the remainder is discarded, thus causing environmental problems (Pesta and others 2007). According to Miller (2003), lactose from whey has increased exponentially in recent years due to the high demand for production of cheese and whey protein concentrate. This has been compounded by low demand and as yet limited applications for lactose. As a result, GOS manufacture from whey lactose is considered a value-adding process and a commercially feasible process (Albayrak and Yang 2002).

Dairy products containing lactose as well as pure lactose solutions are therefore the main substrates from which GOS can be synthesized using β-galactosidase. For example, yogurt can be used as a basic material for the production of GOS via the use of yogurt-based starter cultures during the incubation period. Yogurt containing B. infantis has been reported to have a higher concentration of GOS than yogurt containing other bifidobacteria (Lamoureux and others 2002), thus implying a possible higher transgalactosyl reaction by β-galactosidase from this microorganism.

Galactosyl transferase synthesis using nucleotide phospho-sugars as substrates has also been used to produce GOS (Prenosil and others 1987). In addition, specific enzyme substrates such as o-nitrophenyl galactopyranoside (oNPG) could be used for GOS synthesis using β-D-galactosidase. According to Kim and others (1997), GOSs have been produced from specific substrates enhanced by special ion activators. For example, the authors showed that β-galactosidase from K. lactis were able to produce GOS from oNPG with the addition of Mg2+ and Mn2+. They also reported that the Co2+, Zn2+, and Ni2+ were able to activate the oNPG-hydrolyzing activity of the enzyme while inhibiting the lactose-hydrolyzing ability. Table 1 shows a summary of fungal and bacterial β-galactosidases, their optimum transferase and hydrolytic conditions on lactose, as well as activators and inhibitors. Other activators that are proven to enhance GOS synthesis from lactose include K+, Na+, Fe2+, and ethylene diamine tetraacetic acid (EDTA) also summarized in Table 1. Therefore, what activates the hydrolyzing and transgalactosyl activity of 1 microorganism could be inhibitory to another at different reaction conditions. Thus, the activation-inhibition effect during reactions is specific to a microorganism and also the reaction conditions.

Table 1–.  Characteristics of β-galactosidases from fungal, yeast, and bacterial sources.
OriginpHoptToptLactose Km (mM)M, kDActivatorInhibitorReference
  1. kD = kiloDalton; oNPG = o-nitrophenyl β- D galactopyranoside; SDS = sodium dodecyl sulfate; EDTA = ethylene diamine tetraacetic acid; PCMB = p-chloromercuribenzoate.

Fungal       
Aspergillus niger3.55885124Aehle (2004)
Aspergillus oryzae5.05550 90Aehle (2004)
Yeast       
Kluyveromyces lactis6.53735115K+, Mg2+Ca2+,  Na2+, Zn, CuAehle (2004)
Kluyveromyces fragilis6.6300.23-0.99 (oNPG)200SDSJurado and others (2004)
Sterigmatomyces elviae CBS81196.060Fe2+, Zn2+, Cu2+Onishi and Tanaka (1998)
Bacterial       
Escherichia coli7.240 2540Na+, K+Aehle (2004)
Bacillus subtilis6.550700Aehle (2004)
Bacillus stearothermophilus6.255 2220Mg2+ Aehle (2004)
Lactobacillus thermophilus6.255 6540Aehle (2004)
Lactobacillus reuteri L1038.04513 35Na+, K+, and Mn2+Fe2+, Ca2+, Cu2+, and Zn2+Nguyen and others (2006)
Lactobacillus reuteri L4616.55031 72Na+, K+, and Mn2+Fe2+, Ca2+, Cu2+, and Zn2+Nguyen and others (2006)
Bullera singularis KCTC 7534550580 53Ag3+, SDSCho and others (2003)
Bacillus stearothermophilus7.070 2.96 70Fe2+, Zn2+, Cu2+, Pb2+, and Sn2+Chen and others (2008)
Bifidobacterium bifidum4.845800362EDTAZn2+, Mn2+ Co2+, Ca2+, Sn2+Dumortier and others (1994)
Bifidobacterium infantis5.060 2.6470Na+, K+Cr3+,EDTAc,urea, galactose PCMBHung and Lee (2002)
Bacillus circulans6.06541.7 67Nakanishi and others (1983)
Bacillus coagulans6.55550430EDTAFe3+, Cu2+, Zn2+, and galactoseBatra and others (2005)

GOS Yields Using Different Enzyme Sources

  1. Top of page
  2. Abstract
  3. Introduction
  4. Progress in the Understanding of Transgalactosyl Reactions
  5. Substrate Sources for GOS Synthesis
  6. GOS Yields Using Different Enzyme Sources
  7. Glycosyl-Linkage Analysis of GOSs
  8. Determinants of GOS Composition, DP and Glycosyl Linkages during Synthesis
  9. The Effect of Fermentation Parameters on the Yield of GOS Synthesis
  10. Role of Thermostable Enzymes in GOS Synthesis
  11. Analysis of GOSs
  12. Possible Ways of Improving GOS Yield
  13. Safety Aspects of GOSs
  14. Applications of GOSs as Functional Food Ingredients
  15. Future Prospects
  16. Acknowledgments
  17. References

GOS yield is mainly dependent on reaction factors such as substrate concentration (lactose), reaction temperature, enzyme activity, source of enzyme, and the length of reaction. In general, the yield of GOS would be likely higher with an increase in lactose concentration and vice versa. As shown in Table 2, the influence of β-galactosidases from yeasts and bacteria determines GOS yields. On the other hand, Table 3 highlights the effects of batch and continuous processes on GOS yields. In a continuous process, there is no limitation factor of accumulating end products during a reaction, which favors transgalactosylation and therefore leads to higher GOS yields. The element of shaking or mixing enhances enzyme performance, thus favoring higher GOS yields compared to a batch process in which there is no mixing.

Table 2–.  GOS yield as influenced by purified β-galactosidases from various microorganisms and lactose concentrations.
MicroorganismInitial lactose concentration (g/L)Galactooligosaccharide (conversion yield, ww−1%)Reference
  1. A dash indicates that lactose concentration is not provided in the original referenced source.

Rhodotorula minuta20038%Onishi and Yokozeki (1996)
Sterigmatomyces elviae20039%Onishi and Tanaka (1995)
Sterigmatomyces elviae CBS811936063%Onishi and Tanaka (1998)
Penicillium sp. KFCC 1088840040%In and Chae (1998)
Bacillus circulans 4741%Mozaffar and others (1986)
Saccharopolyspora rectivirgular60044%Nakao and others (1994)
Bullera singularis18050%Cho and others (2003)
Sulfolobus solfataricus48%Reuter and others (1999)
Aspergillus oryzae36%Reuter and others (1999)
Escherichia coli32%Reuter and others (1999)
Aspergillus oryzae22%Matella and others (2006)
Bullera singularis ATCC 2419354%Shin and others (1998)
Lactobacillus reuteri36%Splechtna and others (2007)
Talaromyces thermophilus CBS 23658 8040%Nakkharat and Haltrich (2007)
Thermus sp. Z-1158.440%Akiyama and others (2001)
Table 3–.  Galactooligosaccharide (GOS) yield from lactose solutions by batch and continuous processes.
SourceModeReaction conditionsProductivity (GOS yield)aReference
  1. aGOS yield is a weight percentage of oligosaccharides based on total saccharides in the reaction medium.

  2. SE = soluble enzyme; IE = immobilized enzyme; CSTR = continuous stirred tank reactor; PBR = packed bed reactor.

Aspergillus oryzaeBatch (SE)38% lactose, 40 °C and pH 4.532%Iwasaki and others (1995)
Aspergillus oryzae ATCC 20423Batch (IE)30 ˚C and pH 4.6 Lactose conc. not given26%Sheu and others (1998)
Aspergillus oryzaeContinuous (IE, PBR)4.0% lactose, 40 °C and pH 4.526.6%Albayrak and Yang (2002)
Bacillus circulansBatch (SE)4.56% lactose, 40 °C and pH 6.024%Mozaffar and others (1986)
Bacillus circulansContinuous (IE, CSTR)4.56% lactose, 40 °C and pH 6.040%Mozaffar and others (1986)
Bacillus singularisBatch (IE)30% lactose, 45 °C and pH 3.754%Shin and others (1998)
Bacillus singularisContinuous (IE, PBR)10% lactose, 45 °C and pH 4.855%Shin and others (1998)
Thermus aquaticus YT-1Batch (IE)16% lactose, 70 °C and pH 4.034.8%Berger and Venhaus (1992)
Lactobacillus reuteriBatchNot given36%Splechtna and others (2007)
Lactobacillus reuteriContinuous (CSTR)Not given30%Splechtna and others (2007)
Pyrococcus furiosus (F426Y)Batch70% (wv−1) lactose, 95 °C and pH 5.045%Hansson and others (2001)
Penicillium simplicissimumBatch60% (wv−1) lactose, 50 °C and pH 6.530.5%Cruz and others (1999)
Kluyveromyces lactis (Lactozym 3000 L HP G)Batch25% (wv−1) lactose, 40 °C and pH 6.517.1%Martinez-Villaluenga and others (2008)
Bullera singularis KCTC 7534Batch20% (wv−1) lactose, 50 °C and pH 5.050%Cho and others (2003)
Saccharopolyspora rectivirgulaBatch60% (wv−1) lactose, 70 °C, and pH 6.041%Nakao and others (1994)
Bifidobacterium bifidum BB-12Batch (shaking)5 to 30% (ww−1) lactose, 55°C and pH 7.537.6%Rabiu and others (2001)
Bifidobacterium bifidum NCIMB 41171Continuous45-50% (wv−1) lactose, 40 – 45 °C and pH 6.836 – 43%Goulas and others (2007)
Bifidobacterium angulatumBatch (shaking)5 to 30% (ww−1) lactose, 55 °C and pH 7.543.8%Rabiu and others (2001)
Bifidobacterium infantisBatch (shaking)5 to 30% (ww−1) lactose, 55 °C and pH 7.547.6%Rabiu and others (2001)
Bifidobacterium pseudolongumBatch (shaking)5 to 30% (ww−1) lactose, 55 °C and pH 7.526.8%Rabiu and others (2001)
Bifidobacterium adolescentisBatch (shaking)5 to 30% (ww−1) lactose, 55 °C and pH 7.543.1%Rabiu and others (2001)

In a study by Dumortier and others (1994), 60% of the initial lactose concentration and B. bifidum cells yielded 29% GOS. In previous studies involving wild-type β-glycosidases, transgalactosyl reactions from lactose resulted in maximal GOS yields of 40% to 42% (Nakao and others 1994; Onishi and Tanaka 1995). These yields could represent the upper limit for what is possible to achieve by wild-type β-glycosidases.

Glycosyl-Linkage Analysis of GOSs

  1. Top of page
  2. Abstract
  3. Introduction
  4. Progress in the Understanding of Transgalactosyl Reactions
  5. Substrate Sources for GOS Synthesis
  6. GOS Yields Using Different Enzyme Sources
  7. Glycosyl-Linkage Analysis of GOSs
  8. Determinants of GOS Composition, DP and Glycosyl Linkages during Synthesis
  9. The Effect of Fermentation Parameters on the Yield of GOS Synthesis
  10. Role of Thermostable Enzymes in GOS Synthesis
  11. Analysis of GOSs
  12. Possible Ways of Improving GOS Yield
  13. Safety Aspects of GOSs
  14. Applications of GOSs as Functional Food Ingredients
  15. Future Prospects
  16. Acknowledgments
  17. References

For a complete understanding of the sugar structure, the study should involve aspects such as sugar identification, stereochemistry, types of linkages, ring structures, anomeric configurations, and the sugar sequences in oligosaccharides. Glycosyl linkage analysis, also referred to as methylation analysis, is one of the important procedures in the determination of structural composition of polysaccharides (Rabiu and others 2001). The interpretation of glycosyl linkages is based on electron impact mass spectra of partially O-methylated, and partially O-acetylated alditol derivatives from gas chromatography-mass spectrometry (GC-MS). Briefly, the main steps involve derivatization of the sugars in the samples through partial methylation followed by partial acetylation. The sequential reaction of the sample material with methanolic acid, tert-butanol, and dissolution in methanol leads to the formation of a methyl glycoside. The methyl glycosides are then dissolved in methyl sulfoxide and potassium methylsulfinyl-methanide to deprotonate most of the hydroxyl groups in the sample before addition of iodomethane to obtain a partially methylated methyl glycoside. Acetylation with addition of acetic anhydride is a very slow reaction process, thus requiring the use of 1-methylimidazole as a catalyst. Extraction of the partially methylated, partially acetylated methyl glycoside is achieved through extraction in water and dichloromethane and evaporated in a stream of filtered air to remove excess dichloromethane. The partially methylated, partially acetylated methyl glycosides are then hydrolyzed using trifluoroacetic acid and subsequently reduced by addition of sodium tetradeuteroborate (NaBD4) to form partially methylated alditol acetates (PMAAs). The acetylation of PMAAs is attained by dissolution in glacial acetic acid, ethyl acetate, and acetic anhydride in a specific ratio. The acetylation reaction can be slow, therefore has to be catalyzed by addition of a strong acid such as 60% perchloric acid. After sequential extraction with dichloromethane, the extracted layer is injected for analysis in GLC and GLC-MS. A mass spectrum (spectrum of masses of intact and fragmented original species) of PMAAs is used to identify glycosyl residue linkages. A beam of electrons breaks the PMAAs apart, generating fragment ions in the gas phase and the ions are projected into a mass analyzer, and the mass of the fragments are counted. Thus, it is possible to characterize the GOS synthesized based on galactosyl bond types during transgalactosylation.

Determinants of GOS Composition, DP and Glycosyl Linkages during Synthesis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Progress in the Understanding of Transgalactosyl Reactions
  5. Substrate Sources for GOS Synthesis
  6. GOS Yields Using Different Enzyme Sources
  7. Glycosyl-Linkage Analysis of GOSs
  8. Determinants of GOS Composition, DP and Glycosyl Linkages during Synthesis
  9. The Effect of Fermentation Parameters on the Yield of GOS Synthesis
  10. Role of Thermostable Enzymes in GOS Synthesis
  11. Analysis of GOSs
  12. Possible Ways of Improving GOS Yield
  13. Safety Aspects of GOSs
  14. Applications of GOSs as Functional Food Ingredients
  15. Future Prospects
  16. Acknowledgments
  17. References

From the early 1950s, different chain lengths of oligosaccharides were obtained by β-galactosidases from different microorganisms. For example, 4 different oligosaccharides were obtained using enzyme from K. fragilis, while 3 oligosaccharides were obtained using β-galactosidase from E. coli (Aronson 1952). Pazur (1954) repeated the same experiment using lactase from K. fragilis and found similar results, while others (Roberts and Pettinati 1957; van der Meulen and others 2004) found that by increasing the initial lactose concentration, a much greater variety (11 types) of oligosaccharides was be formed. During transgalactosyl reactions, there are species of oligosaccharides that tend to be dominant in the solution, for example, allolactose as reported in the study by Huber and others (1976). Using β-galactosidase from K. lads on a known lactose solution, Dickson and others (1979) also detected allolactose, galactobiose, and tri- and tetra-oligosaccharides. In the 1980s, Toba and others (1981) also confirmed that β-galactosidase from different microorganisms yielded varying concentrations and compositions of oligosaccharides. In standardized large-scale productions, using the β-galactosidase derived from B. circulans, more than 55% of the lactose was reportedly converted to GOS (Mozaffar and others 1986). Although tri- to hexa-saccharides with 2 to 5 galactose units are the main products of this reaction, disaccharides consisting of galactose and glucose with different β-glycoside bonds from lactose were also produced (Sako and others 1999). In another study by Hsu and others (2007), the production of GOS using β-galactosidase from B. longum BCRC 15708 resulted in 2 types of GOSs, tri and tetra-saccharides, from a lactose concentration of 40% (wv−1). In this study, trisaccharides were the major type of GOS formed. A maximum yield of 32.5% (ww−1) GOS could be achieved from a 40% (wv−1) lactose solution at 45 °C and pH 6.8. Table 4 shows a summary of types of galactosyl linkages formed during transgalactosylation by β-galactosidase from different microorganisms. As shown in Table 4, synthesis of GOS from lactose and whey permeate using the whole cells of B. bifidum NCIMB 41171 gave rise to a variety of oligosaccharides with different degrees of polymerization (DP > 3) and transgalactosylated disaccharides (Goulas and others 2007). Therefore, whereas different microorganisms seemingly yield different compositions of oligosaccharides with varying degrees of polymerization, it appears that the primary determinants are the actual reaction conditions. In addition, for reasons that are still not very clear, certain microorganisms express enzymes that only synthesize certain types of GOS. For example, it has been established that β-galactosidases derived from B. circulans (Mozaffar and others 1986) or Cryptococcus laurentii (Ozawa and others 1989) synthesize mainly β1′4 bonds (4′-GOS) during lactose hydrolysis. Conversely, when enzymes derived from A. oryzae or Streptococcus thermophilus (Matsumoto 1990) were used, β1′6 bonds (6′-GOS) were formed. It is possible that the predominant transgalactosyl enzymes in these microorganisms, owing to their specificity, simply respond to the stereochemical configurations of the glycosyl substrates in the reaction. Therefore, at this point in time, it appears that GOS composition, the galactosyl bond types, and the DP may be determined by a combination of factors including reaction temperature, the purity of the enzyme, pH, initial lactose concentration (water activity), and the source and form of the enzyme. Interestingly, Goulas and others (2007) reported that crude endogenous enzymes produced mixed compositions of GOS, containing both α and β bonds. This scenario is unlikely when pure β-galactosidase or α-galactosidase is used as opposed to crude cellular galactosidase. It is reasonable, however, to expect that pure β-galactosidase would synthesize β-GOS and α-galactosidase (α-GOS).

Table 4–.  Types and degree of polymerization of synthesized galactooligosaccharides produced by different microorganisms.
MicroorganismPolymerization and compositionTypes of linkagesReference
Bifidobacterium bifidum NCIMB 41171DisaccharideGal(α1′6)-GalGoulas and others (2007)
TrisaccharidesGal(β1′6)-Gal(β1′4)-GlcGoulas and others (2007)
 Gal(β1′3)-Gal(β1′4)-Glc 
TetrasaccharideGal(β1′6)-Gal(β1′6)-Gal(β1′4)-GlcGoulas and others (2007)
PentasaccharideGal(β1′6)-Gal(β1′6)- Gal(β1′6)-Gal(β1′4)-GlcGoulas and others (2007)
Bifidobacterium bifidum DSM 20456DisaccharidesGal(β1′6)-GlcDumortier and others (1990)
 Gal(β1′3)-Glc 
 Gal(β1′6)-Gal 
TrisaccharidesGal(β1′3)-Gal(β1′4)-Glc 
 Gal(β1′6)-Gal(βI′4)-Glc 
 Gal(β1′2)-Gal(βI′6)-Glc 
TetrasaccharidesGal(β1′3)-Gal(βl′3)-Gal(βI′4)-Glc 
PentasaccharideGal(βl′3)-Gal(β1′3)-Gal(βI′3)-Gal(β1′4)-Glc 
HexasaccharideGal(βI′3)-Gal(βl′3)-Gal(β1′3)-Gal(βI′3)-Gal(β I′4)-Glc 
HeptasaccharideGal(βl′3)-Gal(β1′3)-Gal(βI′3)-Gal(β1′3)-Gal(βI′3)-Gal(β1′4)-Glc 
Sulfolobus solfataricus (SsbGly)DisaccharideGal(β1′6)-GlcPetzelbauer and others (2000a)
TrisaccharideGal(β1′6)-Gal (β1′6)-Glc 
Pyrococcus furiosus (CelB)DisaccharideGal(β1′3)-GlcPetzelbauer and others (2000a)
TrisaccharideGal(β1′ 3)- Gal(β1′3)-Glc 

The Effect of Fermentation Parameters on the Yield of GOS Synthesis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Progress in the Understanding of Transgalactosyl Reactions
  5. Substrate Sources for GOS Synthesis
  6. GOS Yields Using Different Enzyme Sources
  7. Glycosyl-Linkage Analysis of GOSs
  8. Determinants of GOS Composition, DP and Glycosyl Linkages during Synthesis
  9. The Effect of Fermentation Parameters on the Yield of GOS Synthesis
  10. Role of Thermostable Enzymes in GOS Synthesis
  11. Analysis of GOSs
  12. Possible Ways of Improving GOS Yield
  13. Safety Aspects of GOSs
  14. Applications of GOSs as Functional Food Ingredients
  15. Future Prospects
  16. Acknowledgments
  17. References

Table 3 and 4 show the summary of GOS yields as determined by enzymes from various microorganisms, lactose concentrations, and the influence of the reaction mode. For a long time, the initial lactose concentration has been an important determinant of GOS yield (Wierzbicki and others 1973). Following this finding, Burvall and others (1980) and Asp and others (1980) established that with increased initial lactose concentration a GOS yield of 65-76% (wv−1) could be obtained using enzyme from K. lactis. The information from Table 3 and 4 confirms that microorganisms with high β-galactosidase activity can be important in the synthesis of GOS. Bacterial sources of β-galactosidases can be used as live whole cells (Goulas and others 2007) in which the crude form of the enzyme is able to provide both hydrolytic and transferase activity. Alternatively, these enzymes can be extracted and purified to provide a stable higher activity leading to a higher yield of transgalactosyl products. However, the use of whole-cell and live probiotic microorganisms could provide additional probiotic effect.

The cleavage of the different glycosidic linkages as found in GOS mixtures to varying extents is an important prerequisite for an oligosaccharide mixture to be efficiently utilized by targeted probiotics and hence to exert their specific prebiotic effect (Nguyen and others 2006). It is conceivable that probiotic β-galactosidases, which rapidly hydrolyze certain GOS structures, can preferentially form these glycosidic linkages when acting in the transgalactosylation mode. The use of B. bifidum galactosidases, instead of other galactosidases from different microbial sources, could potentially yield GOSs with improved prebiotic qualities, since the predominant microbial species in the colon are bifidobacteria (Gibson and Roberfroid 1995). As summarized in Table 2, initial lactose concentration also determines GOS yield. The higher the initial lactose concentration, the higher the GOS yield. Higher lactose concentration indicates lower water activity that appears to favor transgalactosylation reaction, while lower lactose concentration, leading to higher water activity, conversely favors hydrolysis. According to the summary in Table 2, Onishi and Yokozeki (1996) achieved a conversion GOS yield of 63% from a 36% lactose concentration as opposed to only 39% GOS yield from 20% lactose concentration. In addition, different microorganisms with varying enzyme activities influence GOS yield, with those having higher enzyme activity yielding higher GOS and vice versa. Initial lactose concentration and the enzyme activity of the microorganism, though, are not the only relevant factors for GOS synthesis. As shown in Table 3, the optimum temperature, pH, and the mode of reaction are critical too in determining GOS yield. For example, according to Shin and others (1998), an immobilized enzyme in a continuously stirred-tank reactor (CSTR) with a lower lactose concentration yielded higher GOS than a batch process with a higher lactose concentration involving the same enzyme extracted from Bacillus singularis. All these parameters influence GOS yield to varying degrees in a reaction process. Table 3, therefore, implies that the reaction during GOS synthesis can be optimized by considering all optimal conditions.

Among different microorganisms, GOSs have been produced by β-galactosidases from A. oryzae (Iwasaki and others 1995), Sirobasidium magnum (Onishi and Tanaka 1998), Penicillium simplicissimum (Cruz and others 1999), B. circulans (Boon and others 1999), B. infantis (Hung and Lee 2002), Bullera singularis and E. coli (Cho and others 2003), and K. lactis (Kim and others 2004). These mesophilic microorganisms are, however, unable to produce GOS at a high temperature, which would be desirable because it enables better control of the solubility of lactose as a substrate, while repressing causes of contamination. Therefore, thermostable β-galactosidases are needed that can produce GOS at high temperatures.

Role of Thermostable Enzymes in GOS Synthesis

  1. Top of page
  2. Abstract
  3. Introduction
  4. Progress in the Understanding of Transgalactosyl Reactions
  5. Substrate Sources for GOS Synthesis
  6. GOS Yields Using Different Enzyme Sources
  7. Glycosyl-Linkage Analysis of GOSs
  8. Determinants of GOS Composition, DP and Glycosyl Linkages during Synthesis
  9. The Effect of Fermentation Parameters on the Yield of GOS Synthesis
  10. Role of Thermostable Enzymes in GOS Synthesis
  11. Analysis of GOSs
  12. Possible Ways of Improving GOS Yield
  13. Safety Aspects of GOSs
  14. Applications of GOSs as Functional Food Ingredients
  15. Future Prospects
  16. Acknowledgments
  17. References

Due to their superior stability properties, glycosidases from thermophilic microorganisms have become an attractive interest as an alternative to the nonthermostable enzymes. Consequently, several researchers have studied thermostable microorganisms such as Thermus sp. Z-1 (Akiyama and others 2001), Pyrococcus furiosus (Bruins and others 2003), S. elviae (Onishi and Tanaka 1995), Rhodotorula minuta (Onishi and Yokozeki 1996), and Sulfolobus solfataricus (Reuter and others 1999) in search of GOS production at high temperatures. The rationale for the significance of using thermostable enzymes is that transgalactosyl reactions proceed more efficiently at high substrate concentrations. For GOS production, the main benefit of using higher reaction temperatures lies in the increase of lactose concentration that improves GOS yield. Lactose generally has low solubility at lower temperatures and this limits the reaction leading to low GOS yield. Therefore, the possibility of developing new enzymes that act at higher temperatures at which the solubility of lactose is increased is a significant step toward increase in GOS yield. Also, thermostable enzymes have increased stability at elevated temperatures, limiting risks of microbial contamination (Gekas and Lopez-Leiva 1985), lower viscosity, improved transfer rates, and improved solubility of substrates and products. However, possible limitations in the use of thermozymes may be found in the instability of co-factors, substrates or products, and the occurrence of unwanted side reactions such as Maillard that might lead to enzyme inactivation during the reaction. Mesophilic β-glycosidases (Mahoney 1998) and thermophilic ones (Sako and others 1999) are not very stable in a thermal reactor over a longer time in a commercial setting. This affects the stability of the enzyme, consequently affecting negatively the GOS yield. Therefore, the operational high temperature presents a compromise between reasonable stabilities of the enzymes and the occurrence of unwanted browning brought about by the Maillard reaction (Petzelbauer and others 1999).

More recently, a thermostable β-galactosidase gene bgaB from Bacillus stearothermophilus was cloned and expressed in B. subtilis WB600 (Chen and others 2008). The optimum temperature and pH for its β-galactosidase activity were 70 °C and pH 7.0, respectively. This enzyme was found to possess a high level of transgalactosylation activity in the hydrolysis of lactose in milk. The results suggest that this recombinant thermostable enzyme may be suitable for both the hydrolysis of lactose and the production of GOS in milk processing (Chen and others 2008). A few papers have also described GOS production using thermostable recombinant β-galactosidases from hyperthermophilic bacteria. An example of a thermostable recombinant microorganism is Thermotoga maritima expressed in E. coli whose maximal GOS production was at pH 6.0 and 90 ˚C. These recombinant enzymes have some advantages in comparison with native enzymes, since the recombinant ones are easy to purify and large-scale production is easily achievable compared to native ones (Ji and others 2005). Recombinant enzymes are also more stable at higher temperatures than the corresponding native counterparts. Therefore, genetic engineering remains a viable option for synthesis of higher GOS yields. A more efficient GOS production of high yield, with genetic engineering, could be an important option for commercial application (Onishi and Tanaka 1998).

Lactose hydrolysis using extremely thermostable β-glycosidases at high temperatures of between 65 °C and 75 °C by hydrolytic thermozymes has been specifically conducted by Petzelbauer and others (1999, 2000a). At the high temperature of more than 70 °C, transgalactosylation was reported to compete with hydrolysis during lactose conversion using β-glycosidases from S. solfataricus (SsbGly) and P. furiosus (CelB) (Petzelbauer and others 2000a). Previous studies by the same group had shown that both SsbGly and CelB are useful biocatalysts for lactose conversion at this high reaction temperature (Petzelbauer and others 1999).

Analysis of GOSs

  1. Top of page
  2. Abstract
  3. Introduction
  4. Progress in the Understanding of Transgalactosyl Reactions
  5. Substrate Sources for GOS Synthesis
  6. GOS Yields Using Different Enzyme Sources
  7. Glycosyl-Linkage Analysis of GOSs
  8. Determinants of GOS Composition, DP and Glycosyl Linkages during Synthesis
  9. The Effect of Fermentation Parameters on the Yield of GOS Synthesis
  10. Role of Thermostable Enzymes in GOS Synthesis
  11. Analysis of GOSs
  12. Possible Ways of Improving GOS Yield
  13. Safety Aspects of GOSs
  14. Applications of GOSs as Functional Food Ingredients
  15. Future Prospects
  16. Acknowledgments
  17. References

Liquid chromatography has been largely used depending on the matrix from which GOS is to be extracted and analyzed. Suitable types of saccharide HPLC columns and detectors, usually refractive index (RID), have also been employed along with appropriate analytical conditions. However, when it comes to analysis of GOS in dairy products such as in milk, skim milk, and milk with high solids levels, very little has been accomplished due to the presence of casein and whey proteins.

It has been suggested that the use of carrez reagents (potassium hexacyanoferrate(II) 3-hydrate, potassium ferrocyanide, and zinc sulfate) and perchloric acid might be useful in protein clarification and precipitation from dairy-based matrices to overcome inherent obstacles in GOS analysis from these products (Cabálková and others 2004). Carrez reagents present problems with residual proteins that would affect column operation during HPLC analysis, while perchloric acid creates a highly acidic medium that eventually requires neutralization with a strong alkali, thus complicating further the analytical process. Ion exchange could be a possibility in eliminating perchlorate ions. However, methanol–chloroform extraction remains a more suitable alternative of protein removal thus precluding the need for the use of perchloric acid.

Still then, the most practical and accurate analytical means of identifying and quantifying individual GOS synthesized in dairy products is the high-performance liquid chromatography (HPLC). Quemener and others (1997) developed a method based on high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) to measure GOS in food and feed products. A few years later, de Slegte (2002) organized a successful Assn. of Official Analytical Chemists (AOAC) collaborative study of this method in which galactose and other sugars were separated on a CarboPacTM PA1 column and detected by pulsed amperometric detection (PAD) using a triple potential waveform. HPAE-PAD has been found to be more superior in the detection of GOSs than high-performance liquid chromatography with RI detection. However, in the event that HPAE-PAD is not available for use, HPLC-RI can be reliably used instead. Although the RI detector has several limitations, namely the dependence of sensitivity on changes in solvent composition, temperature, and pressure, it however remains the most useful tool so far in the determination of sugar concentrations in foods. Detection limits of substances using HPLC in general are largely dependent on the compound being analyzed as well as the sensitivity of detector used. Whereas the RI detector is not the most sensitive, for example being 100 to 1000 less sensitive than UV detectors, its suitability with detection range of micrograms per milliliter (μg/mL) is convenient enough for GOS detection and analysis.

Possible Ways of Improving GOS Yield

  1. Top of page
  2. Abstract
  3. Introduction
  4. Progress in the Understanding of Transgalactosyl Reactions
  5. Substrate Sources for GOS Synthesis
  6. GOS Yields Using Different Enzyme Sources
  7. Glycosyl-Linkage Analysis of GOSs
  8. Determinants of GOS Composition, DP and Glycosyl Linkages during Synthesis
  9. The Effect of Fermentation Parameters on the Yield of GOS Synthesis
  10. Role of Thermostable Enzymes in GOS Synthesis
  11. Analysis of GOSs
  12. Possible Ways of Improving GOS Yield
  13. Safety Aspects of GOSs
  14. Applications of GOSs as Functional Food Ingredients
  15. Future Prospects
  16. Acknowledgments
  17. References

In any commercial enzyme process, it is crucial to separate valuable enzyme from the product stream for reuse with a fresh substrate. Ultrafiltration (UF) is a process where fluid containing enzyme and product flow at a high rate across a membrane surface at a certain fluid pressure. Table 5 shows the effect of UF on GOS purification based on initial lactose concentration and temperature. Depending on membrane-pore size, the enzyme is retained while smaller chemicals (that is, sugars such as GOS) are permeated (Matella and others 2006). Foda and Lopez-Leiva (2000) reported that UF technology in a continuous free-enzyme system can produce GOS from whey permeate. The study by (Matella and others 2006) showed that fluid pressure effects on enzyme are negligible in free enzyme UF systems and that increased agitation causes enhanced solubility of enzymes and sugars thereby increasing enzyme performance. In their study, compatible free- and immobilized- enzyme recycle batch systems showed approximately 20% to 22% maximum GOS production within 15 to 17 minutes. Thus, a continuous UF system may be 1 way of improving GOS yield.

Table 5–.  The effect of ultrafiltration on galactooligosaccharide (GOS) yield at different temperatures.
ModeLactose concentrationTemperatureGOS yieldaReferences
  1. aInitial GOS yield before UF was not provided in the referenced sources.

Ultrafiltration (UF)100 g/L30 ˚C15%Sheu and others (1998)
Ultrafiltration (UF)270 g/L40 ˚C20-22%Matella and others (2006)

Figure 2 shows a schematic process of a continuous UF membrane bioreactor in which enzyme performance can be enhanced, leading to higher GOS yields. With a given reactor volume, the feed flow rate can be controlled by an electronic diaphragm pump, which could be adjusted to work under a set bar pressure by means of the restriction valve. In the work conducted by Chockchaisawasdee and others (2004) a volume of filtered lactose solution was heated and the enzyme Maxilact® L2000 was added to initiate the reaction. The reactor was stirred to ensure adequate mixing of enzyme and substrate. During the synthesis, fresh lactose substrate was fed into the reactor to replace the volume of permeate stream removed. The volume of permeate can be recorded and samples collected at specific time intervals for GOS analysis. A batch synthesis of GOS with a similar lactose concentration and similar reaction conditions can be performed to compare the effectiveness of the 2 systems on GOS yield. UF can therefore be 1 way of increasing GOS yield from low molecular weight sugars based on size-exclusion technique.

image

Figure 2–. Ultrafiltration membrane bioreactor: (1) feed, (2) reactor vessel, (3) magnetic bar, (4) electronic diaphragm pump with adjustable speed, (5) inlet pressure gauge, (6) the Pellicon XL device containing ultrafiltration membrane, (7) outlet (retentate) pressure gauge, (8) restriction valve for pressure adjustment, (9) permeate stream, (10) air bath.

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Temperature also influences GOS yield by determining the speed of molecular interaction during reaction, the activation energy, and the thermal stability of the enzyme and substrate. As a result, a reaction process at the optimum temperature leads to synthesis of higher GOS yield. For this reason, thermostable enzymes are likely to synthesize higher GOS yields at higher temperatures than at a lower temperature. Thus, in the 2 studies compared, as is shown in Table 5, a 5% to 7% higher GOS yield was achieved at a reaction temperature of 40 °C than at 30 °C.

Another way of improving GOS yield is by microwave irradiation. Maugard and others (2003) were able to establish that microwave irradiation stimulated GOS production using β-glycosidases from K. lactis. Microwave affects enzymatic properties such as enzyme activity, selectivity, and stability. The influence of microwave irradiation on enzymatic activity is due to the hydration state and the polarity of the reaction medium. During irradiation, heating occurs and in an open system, microwave exposure induces an equilibrium shift by evaporation of light polar molecules (for example, water) which strongly react with the electromagnetic field. At high temperatures in an open system, evaporation can cause a reduction on enzyme activity. However, in a closed system, no evaporation occurs, hence the enzyme activity remains intact, but is usually higher than conventional heating at the same reaction temperature. Second, the polarity of the reaction medium also affects enzymatic activity during microwave irradiation (Rejasse and others 2007). It has been observed that enzymatic reaction increases with the changing hydrophobicity of the reaction solvents (Yadav and Lathi 2004). When transesterification or transgalactosyl activity occurs during a reaction, product chain length increases and this influences the medium hydrophobicity. When the medium becomes more hydrophobic, the enzyme activity increases and hence also GOS yield in a transgalactosyl reaction. Microwave heating could also have an effect on the stereoselectivity of the enzyme. The mechanism is still not quite understood, but it is a known fact under certain reaction conditions, preferential formation of 1 stereoisomer over another may occur in a chemical reaction. Finally, microwave irradiation also influences enzyme stability. It is possible that there is a specific effect of microwave irradiation on the structural and functional properties of enzymes. A direct energy transfer during irradiation between the electromagnetic field and the polar proteins's domains could lead to modification of the enzyme's flexibility and, consequently, alter enzymatic properties (La Cara and others 1999). Moreover, direct absorption of the microwave energy by the polar substrates of the enzyme could lead to a higher reactivity of the functional groups involved in the enzymatic reaction (Mazumder and others 2004), thus leading to higher product yield.

Other approaches to increase the production of GOS have been attempted through protein engineering technology and changes in specific amino acids at the active site of the enzyme (Hansson and others 2001). The process by which site-directed mutagenesis occurs in the active sites of an enzyme is referred to as protein engineering. For example, the synthetic properties of a CelB mutant from hyperthermophilic P. furiosus was reportedly enhanced by altering 1 amino acid, phenylalanine to tyrosine (F426Y) in its active site, resulting in a GOS yield of 45% from a 10% lactose solution (Table 3). Compared to the wild type, from the same substrate concentration, GOS yield was only 18%. At this lactose concentration, A. oryzae, β-galactosidase and Caldocellum saccharolyticum β-glycosidase provided oligosaccharide yields of 10% and 7%, respectively (Stevenson and others 1996). For the mutant, the result indicates that the improved yield is due to an increase in the ratio of transglycosylation to hydrolysis. This ratio is determined by the reaction kinetics, hence additional protein engineering of this or other glycosidases could increase oligosaccharide yields. Thus by altering the protein type in the active site of the enzyme, the transgalactosylation activity can be improved leading to an increase in GOS yield.

Safety Aspects of GOSs

  1. Top of page
  2. Abstract
  3. Introduction
  4. Progress in the Understanding of Transgalactosyl Reactions
  5. Substrate Sources for GOS Synthesis
  6. GOS Yields Using Different Enzyme Sources
  7. Glycosyl-Linkage Analysis of GOSs
  8. Determinants of GOS Composition, DP and Glycosyl Linkages during Synthesis
  9. The Effect of Fermentation Parameters on the Yield of GOS Synthesis
  10. Role of Thermostable Enzymes in GOS Synthesis
  11. Analysis of GOSs
  12. Possible Ways of Improving GOS Yield
  13. Safety Aspects of GOSs
  14. Applications of GOSs as Functional Food Ingredients
  15. Future Prospects
  16. Acknowledgments
  17. References

Given that GOSs are synthesized from milk sugar and traditional dairy foods, they are generally recognized as safe (GRAS). Because of their prebiotic potential, they can be important in enhancing intestinal health. In vivo studies in rats indicated that oral administration of GOS at a rate of 20 g/kg body weight once and daily intake at a rate of 1.5 g/kg body weight for 6 months, respectively, showed no toxicity (Sako and others 1999).

Beyond consumption of such high levels, the only known adverse effect of GOS so far is transient diarrhea due to the so-called “osmotic diarrhea.” Threshold GOS amounts that do not induce osmotic diarrhea are approximately estimated to be 0.3 to 0.4 g/kg body weight, an equivalent of about 20 g per average human body (Sako and others 1999). In comparison to animal studies, the use of high levels of GOS resulted in no adverse side effects, such as diarrhea and flatulence, compared with equivalent levels of FOS (Propst and others 2003).

Applications of GOSs as Functional Food Ingredients

  1. Top of page
  2. Abstract
  3. Introduction
  4. Progress in the Understanding of Transgalactosyl Reactions
  5. Substrate Sources for GOS Synthesis
  6. GOS Yields Using Different Enzyme Sources
  7. Glycosyl-Linkage Analysis of GOSs
  8. Determinants of GOS Composition, DP and Glycosyl Linkages during Synthesis
  9. The Effect of Fermentation Parameters on the Yield of GOS Synthesis
  10. Role of Thermostable Enzymes in GOS Synthesis
  11. Analysis of GOSs
  12. Possible Ways of Improving GOS Yield
  13. Safety Aspects of GOSs
  14. Applications of GOSs as Functional Food Ingredients
  15. Future Prospects
  16. Acknowledgments
  17. References

From the very beginning, lactose utilization could only be enhanced by hydrolytic reaction into the monomer sugar components of D-glucose and D-galactose, due to the fact that 70% of the world population is lactose-intolerant (Harju 1987). Lactose-intolerant individuals experience gastrointestinal complications upon consumption of lactose due to lack of lactose-hydrolyzing enzyme (Scrimshaw and Murray 1988). D-glucose and D-galactose are comparatively sweeter and more easily digested than lactose.

Any substrate such as lactose or purified whey can be used in the industrial production of GOS following, as an example, the schematic process shown in Figure 3. β-Galactosidase from a given source may be used to synthesize GOS under optimized conditions followed by the unit operations as shown in the schematic diagram. As shown, GOS manufacture begins with the substrate lactose that maybe from any source or form and must involve a transgalactosyl reaction via a β-galactosidase. This leads to formation of GOS that may involve different linkage types and degrees of polymerization depending on the enzyme source. Often there may be discolorization as a result of side reactions such as Maillard reactions, especially when reactions occur at elevated temperatures. These off-colors may be eliminated by food grade decolorizers. GOS synthesized from dairy-based sources often contain calcium that is beneficial for bone health. However, even these may be removed so as to enhance purity of the products. Filtration follows based on molecular sizes and often involves UF procedures. The filtered liquor is concentrated via evaporation, from which GOS syrup is obtained or can be freeze-dried to obtain GOS powder. The main products from this process usually are trisaccharides, namely β1′4- or β1′6 galactosyllactose, and longer oligosaccharides consisting of 4 or more monosaccharide units. According to Matsumoto and others (1990), substantial amounts of transgalactosylated disaccharides (TD) are also produced in these reactions. Compared to lactose and other larger sugar molecules, GOSs have been found to have low cariogenicity, low caloric values, and low sweetness, thus making them suitable for use as functional ingredients. This makes them great candidates for enhancing the growing number of functional foods.

image

Figure 3–. Scheme of process steps involved in the industrial production of galactooligosaccharides.

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GOSs also have preferable physicochemical characteristics, including stability in acidic and high-temperature conditions. Therefore, they are suitable candidates for supplementation in processed foods such as bread. In bread, a GOS can provide excellent taste and texture (Sako and others 1999) and it can also be quite stable at room temperature even in acidic conditions for a long time. It has been suggested that GOS stability is better than that of FOS (Voragen 1998).

Other processed food products that are important for potential GOS inclusion are fermented dairy products, jams, confectionery, and beverages (Sako and others 1999). Some European countries and Japan already have fermented dairy products containing GOS. An example is Oligomate 55, shown in Table 6, which contains more than 55% of β1′4-GOS and some disaccharides. Oligomate 55 solution is more viscous and has a higher moisture retention capacity compared to high-fructose corn syrup (HFCS), and therefore can better enhance food preservation (Ito and others 1990). Due to the prebiotic effects of GOS, baby foods and specialized foods for the elderly remain important outlets for GOS inclusion. A summary of the commercial GOS products currently available in the market as well as the composition of polymers are shown in Table 6.

Table 6–.  Commercially known galactooligosaccharide-based products currently in the market.
Commercial GOSManufacturerEnzyme sourcePolymerization and compositionReference
  1. aCommercial source not specified in the original reference.

1.Transgalactosylated oligosaccharidesaβ-Galactosidases from Aspergillus oryzaeTri-, tetra-, penta-, and hexa-galactooligosaccharidesTanaka and others (1983)
2. Oligomate 55Yakult (Tokyo, Japan)β-Galactosidases from Aspergillus oryzae and Streptococcus thermophilus36% tri-, tetra-, penta-, and hexa-galactooligosaccharides, 16% disaccharides galactosyl glucose and galactosyl galactose, 38% monosaccharides and 10% lactoseIto and others (1990)
3. Transgalactosylated disaccharideaβ-Galactosidases from Streptococcus thermophilusA mixture of sugars, galactosyl galactose, and galactosyl glucoseIto and others (1993)
4. Vivinal®Borculo Domo Ingredients (Zwolle, The Netherlands).β-Galactosidases from Kluyveromyces lactisTri- and tetra- galactooligosaccharides, lactose, glucose, and galactoseChockchaisawasdee and others (2004)

Future Prospects

  1. Top of page
  2. Abstract
  3. Introduction
  4. Progress in the Understanding of Transgalactosyl Reactions
  5. Substrate Sources for GOS Synthesis
  6. GOS Yields Using Different Enzyme Sources
  7. Glycosyl-Linkage Analysis of GOSs
  8. Determinants of GOS Composition, DP and Glycosyl Linkages during Synthesis
  9. The Effect of Fermentation Parameters on the Yield of GOS Synthesis
  10. Role of Thermostable Enzymes in GOS Synthesis
  11. Analysis of GOSs
  12. Possible Ways of Improving GOS Yield
  13. Safety Aspects of GOSs
  14. Applications of GOSs as Functional Food Ingredients
  15. Future Prospects
  16. Acknowledgments
  17. References

The synthesis of GOS using exogenous β-galactosidase in dairy products is a promising venture for the production of novel functional ingredients important for the health of millions of health-conscious consumers around the world. Even more promising is the fact that these ingredients can be obtained from low-cost substrates such as lactose from whey. Successful analytical processes will allow for targeted synthesis of GOS preferentially metabolized by probiotic gut bacteria to enhance consumer health. The global market for nutraceutical foods such as GOS-based ingredients is sizable and growing and this trend represents an opportunity to create high-value products.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Progress in the Understanding of Transgalactosyl Reactions
  5. Substrate Sources for GOS Synthesis
  6. GOS Yields Using Different Enzyme Sources
  7. Glycosyl-Linkage Analysis of GOSs
  8. Determinants of GOS Composition, DP and Glycosyl Linkages during Synthesis
  9. The Effect of Fermentation Parameters on the Yield of GOS Synthesis
  10. Role of Thermostable Enzymes in GOS Synthesis
  11. Analysis of GOSs
  12. Possible Ways of Improving GOS Yield
  13. Safety Aspects of GOSs
  14. Applications of GOSs as Functional Food Ingredients
  15. Future Prospects
  16. Acknowledgments
  17. References
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