Galactooligosaccharides: Novel Components of Designer Foods
Abstract: Since introduction of functional foods, commercialization of the traditionally used probiotics has ushered in more followers into the new fraternity of sophisticated, health-conscious consumers. In 1995, this was followed by the first introduction of prebiotics. Prebiotics are defined as “a non-digestible feed supplement, beneficially affecting the host by selectively stimulating growth and/or activity in one or a limited number of bacteria in the colon.” The number of new product introductions with prebiotics has steeply increased over the last few years. Paradoxically, probiotics have limited applications as these cannot be used in wide range of food products because of their viability issue. Fortunately, prebiotics do not suffer from any such constraint and can be used in a wide range of food products. Probiotics do not have a long shelf life in their active form. In most cases, refrigeration is required to maintain the shelf life. While probiotics are predominantly used in fermented dairy products, the use of prebiotics has expanded into other food categories. Prebiotics have successfully been incorporated in a wide variety of human food products such as baked goods, sweeteners, yoghurts, nutrition bars, and meal replacement shakes. For instance, the introduction of galacto-oligosaccharides (GOS) into baby foods has been very successful. GOS, which are identical to the human milk oligosaccharides, has emerged with strong clinical support for both digestive and immune health. Various aspects related to GOS such as types and functions of functional food constituents with special reference to GOS, their role as prebiotics, and enhanced industrial production through microbial intervention are dealt in this review.
In older days, food used to be considered as a substance or material eaten or drunk to provide nutritional support for the body or for pleasure. But now the concept of food is changing and foods are intended not to only satisfy hunger and to provide necessary nutrients for humans but also to prevent nutrition-related diseases and improve physical and mental well-being of the consumers (Menrad, 2003). Various researches have demonstrated that there is a delicate balance between the health and the intestinal microflora of the host. It is therefore important that gut microflora interactions should be controlled and sustained in an optimal manner. The concept of functional foods is moving toward the development of food products that beneficially influence the intestinal microbiota. A food can be made functional by increasing the concentration, adding or improving the bioavailability of a particular component. Different types of compounds of plants and animal origin are being used as a component of functional foods. Among the functional components, probiotics, prebiotics, soluble fiber, omega-3—polyunsaturated fatty acids, conjugated linoleic acid, plant antioxidants, vitamins and minerals, some proteins, peptides, and amino acids, as well as phospholipids are frequently mentioned. At the moment, the most important and the most frequently used functional food compounds for gut health are probiotics and prebiotics. Both are rapidly gaining scientific popularity as safe and effective agents that help regulate the body's micro-environment. Paradoxically, probiotics have limited applications as these cannot be used in wide range of food products because of their viability issue. Fortunately, prebiotics do not suffer from any such constraint and can be used in a wide range of food products. Probiotics do not have a long shelf life in their active form. In most cases, refrigeration is required to maintain the shelf life. While probiotics are predominantly used in fermented dairy products, the use of prebiotics has expanded into other food categories. Prebiotics have successfully been incorporated in a wide variety of human food products such as baked goods, sweeteners, yoghurts, nutrition bars, and meal replacement shakes. Oligosaccharides such as inulin and fructooligosaccharides (FOS) are well known for their contribution to digestive health. Another oligosaccharide, galactooligosaccharides (GOS), has also emerged with strong clinical support for both digestive and immune health. In fact, GOS is 1 of only 3 prebiotics recognized by experts for its breadth of clinical evidence. As a stable, soluble ingredient, GOS is an ideal choice for formulating foods and beverages for digestive and immune health. Owing to its similarity to the human milk oligosaccharides (HMO), GOS have attracted worldwide a great of attention of researchers.
The term “Functional Foods” was first introduced in Japan in the mid-1980s and can be defined as a food that is similar in appearance to a conventional food, consumed as part of the usual diet, with demonstrated physiological benefits, and/or to reduce the risk of chronic disease beyond basic nutritional functions (Zheng and others 2006, Health Canada website: http://hc-sc.gc.ca). Because of the absence of an universally accepted term for functional foods, a variety of terms have appeared and are being used worldwide such as designer foods, nutraceuticals, medifoods, vitafoods, and the more traditional dietary supplements and fortified foods.
Prebiotics or Probiotics?
One of the main differences between probiotics and prebiotics is that probiotics are viable food components whereas prebiotics are nonviable food component. Use of probiotics is a way to replenish bacteria levels in the gut with external microorganisms. Food products having probiotic components may contain bacteria that are not necessarily indigenous to the human gut so once in the gut they have to compete to find a place among established, colonized bacteria. Probiotics can also be destroyed by the contents of the gastrointestinal tract. So probiotics cannot be used in wide range of food products because of their viability issue.
On the other hand, prebiotics are nondigestible, remain intact through the digestive system and act as food for already established microflora. The major applications for probiotics are in dairy foods. Prebiotics are added to dairy products, table spreads, baked goods, and breads, breakfast cereals and bars, salad dressings, meat products, and some confectionery items.
So prebiotics overcome many of the traditional limitations of introducing probiotic bacteria in to the GI tract. Therefore, using prebiotics is arguably a more practical and efficient way to manipulate the gut microflora.
The contribution of inulin and FOS to the digestive health is well documented. Another oligosaccharide, GOS has also emerged with strong clinical support for both digestive and immune health. In fact, GOS is 1 of only 3 prebiotics recognized by experts for its breadth of clinical evidence (Gibson and others 2004). As a stable, soluble ingredient, GOS is an ideal choice for formulating foods and beverages for digestive and immune health. Owing to its similarity to the HMO, GOS have attracted worldwide attention of researchers. GOS is unique compared to other prebiotics due to its multi-mechanistic way in which it influences the large intestinal microflora. All prebiotics selectively enhance the growth and/or activity of healthy bacteria but GOS along with this also acts as a receptor decoy to help to inhibit adhesion of certain pathogenic bacteria to the cells that line the large intestine and protect the body from invasion by harmful bacteria. Notably GOS had the highest antiadhesion ability of all prebiotics tested (Shoaf and others 2006).
GOS are nondigestible, carbohydrate-based food ingredients that can enhance health related physiological activities (production of short chain fatty acids [SCFA], energy transduction in colonocytes, growth, and cellular differentiation of colonic epithelial cells, lipid, and carbohydrate metabolism), which can provide protection from infection; decrease the number of potentially pathogenic bacteria; facilitate the normal functions of the gut; stimulate the absorption of some minerals and decrease blood lipids content (Broek and others 2008). GOS, also known as oligogalactosyllactose, oligogalactose, oligolactose, or transgalactooligosaccharides (TOS), belong, because of their indigestible nature, to the group of prebiotics. Because of their known health benefits, GOS have become the centre of a great deal of attention in the field of functional foods. In the past, GOS were acknowledged as food components of little significance due to, among others, their low sweetness, poor solubility in water, and low digestibility. Currently, GOS are claimed to belong to prebiotics, that is, components selectively stimulating the growth of beneficial bacteria in the lower part of the human intestine (Schaafsma 2008).
GOS can be synthesized from 2 sources: soya beans and lactose (from cow's milk). The latter resemble those oligosaccharides that occur naturally in human breast milk and therefore their primary use has so far been in formulations for the infant formula market. The origin of commercial interest in GOS ingredients is mainly because of the presence of galactose-based oligosaccharides in human milk and the structural similarity between commercial GOS and HMO. Human milk contains about 7% lactose and 1% HMOs consisting of lactose with linked fucose, N-acetylglucosamine, and sialic acid. HMO represents the 3rd largest solid component in human milk, following lactose and fat. Most of the previous studies reported the oligosaccharide in human milk consisting of approximately 60% to 90% GOS and 10% to 40% (FOS) in the first few months of lactation (Xiao-ming and others 2004). They primarily support the growth and development of the infant's microflora and immune system (Boehm and others 2005). Human-milk fed babies have higher bifidobacteria levels, less complex microflora composition, and lower susceptibility to infectious disease than formula-fed babies. Higher levels of ammonia, amines, and phenols and other potentially harmful substances have also been found in babies fed powdered milk products (Heavey and others 2003). However, supplementing the proper amount of commercial infant formula with GOS restores levels to those of human-milk fed babies (Bruzzese and others 2006).
Production of GOS
Simply put, GOS are not digested by humans or other animals, and selectively increase the beneficial microflora of the intestine, leading to health benefits that are extensively recognized (Macfarlane and others 2008).
GOS molecules (for example, Gal [β 1→4] Gal [β 1→4] Glc) are typically synthesized by the enzymatic activity of β-galactosidase on lactose in a reaction known as transgalactosylation. Other carbohydrate modifying enzymes, such as β-glucosidases and β-glycosidases, can also catalyze this chemistry. β-Galactosidase belongs to a class of hydrolytic enzymes and has long been used in the dairy industry to hydrolyze lactose, producing glucose and galactose. This hydrolytic activity increases the sweetness of a dairy product, and also lowers lactose concentration, which can be beneficial to lactose intolerant consumers (Lomer and others 2008). But this hydrolytic activity is detrimental for the production of GOS by changing the reaction conditions β-galactosidase is made to catalyze transgalactosylation reaction.
The first event during β-galactosidase activity is the docking of a substrate molecule (Figure 1). When the substrate is lactose the glycan moiety is galactosyl and the aglycan moiety is glucosyl. After the substrate has docked in the active site of the β-galactosidase, catalysis can occur. A covalent bond forms between the galactosyl moiety and the enzyme, followed by galactosyl transfer to an acceptor nucleophile.
The reaction pathway diverges at this point. The result of the reaction depends on the identity of the galactosyl acceptor, for which there is broad specificity. When the galactosyl acceptor is water, free galactose is released. The result of the reaction is hydrolysis of the galactoside, leading to degradation of both lactose and GOS. If the acceptor is a saccharide, GOS is formed; failing that hydrolysis occurs (Gosling and others 2010). Many studies of recombinant thermostable β-galactosidases have been conducted in pursuit of galacto-oligosaccharide production at high temperatures. However, only a few studies have described galacto-oligosaccharide production using recombinant thermostable β-galactosidases, such as those obtained from Geobacillus stearothermophilus (Placier and others 2009), Pyrococcus furiosus (Bruins and others 2003), S. solfataricus (Park and others 2008), T. maritima (Ji and others 2005), and Thermus spp. (Akiyama and others 2001). These recombinant enzymes have several advantages over native enzymes, including ease of purification, large-scale production, and improvements in activity. When the amino acid of the recombinant β-galactosidase from G. stearothermophilus is altered from Arg109 to Trp109, the GOS yield from lactose and the productivity of GOS increase by approximately 12- and 17-fold, respectively (Placier and others 2009).
Generally, as whole cells were utilized, the production rate of GOS is increased by toluene treatment, which increases cell permeability. To obtain a higher of GOS yield from lactose, glucose oxidase and catalase is added to the toluene-treated cells. These enzymes are used to remove glucose as a byproduct, which inhibits the production of GOS. As a result, the yield and productivity of GOS can be increased by 29% and 1.8-fold, respectively (Onishi and others 1996).
Health Benefits of GOS
GOS provide their health benefits by 2 main mechanisms, one is by selective proliferation of beneficial bacteria especially bifidobacteria and lactobacilli in the gut, which provide resistance against colonization of pathogens thereby reducing exogenous and endogenous intestinal infections. These beneficial organisms modulate the immune system and suppress IBD inflammation. The other mechanism is by production of SCFA. Metabolism of GOS leads to the production of SCFA. These SCFA show various beneficial effects including reduction of cancer risk, increase in mineral absorption, improvement in bowel habit, control of serum lipid and cholesterol level, and reduce cancer risk and IBD inflammation (Table 1).
Table 1–. Health benefits of GOS.
|GOS||Humans||GOS induced a modest, but statistically significant increase of bifidobacteria compared to the control treatment||Davis and others (2010)|
|GOS/FOS Saccharomyces boulardii||Rats||IGF-2 levels (ng/mg protein) were significantly elevated in the prebiotics and synbiotic group. Upregulation of Superoxide Dismutase-1 and glutathione peroxidase-3 were observed in the groups having prebiotic diet as compared to the formula-fed diet.||D'souza and others (2010)|
|GOS||Mice||No detectable effect on the intestine villus height, total protein content, and sucrase activity were found to be increased, an increase in O-linked glycoproteins associated with the intestinal mucosa was also observed.||Leforestier and others (2009)|
|GOS (formula food supplemented with GOS)||Human||GOS group had a significantly higher content of fecal bifidobacteria compared with the control group||Fanaro and others (2009)|
|Formula food supplemented with GOS||Human||At the end of a 3-mo feeding period, the number of intestinal bifidobacteria and lactobacilli was significantly increased both in GOS-supplemented formula-fed infants and in breast-fed infants, compared with those fed with the control formula. No difference was seen between the GOS formula-feeding and breast-feeding groups.||Ben and others (2008)|
|B-GOS (Bimuno GOS)||Human||B-GOS had a significant effect on all bacterial groups measured compared with the placebo groups. Higher numbers of Bifidobacterium spp., Lactobacillus-Enterococcus spp., and the C. coccoides–E. rectale group were observed compared with the placebo treatment. In contrast, numbers of Bacteroides spp., C. histolyticum group, E. coli, and Desulfovibrio spp. decreased compared with the placebo treatment.||Vulevic and others (2008)|
|GOS||Rats||Rats on the GOS diet efficiently absorbed Ca and Mg.||Chonan and others (2001)|
|Formula food supplemented mixture of GOS:FOS (9:1)||Human||Number of bifidobacteria greatly increased from initially low levels but no significant effects was found in the level of bacteroides, clostridia, enterobacteria, or yeasts||Boehm and others (2005)|
|V-GOS (Vivinal GOS) B-GOS||Human||Feeding of both B-GOS and V-GOS increase the bifidobacterial population.||Depeint and others (2008)|
|V-GOS||Human||No significant change in serum lipids or glucose absorption were observed.||Van Dokkum and others (1999)|
|GOS||Human||Administration of GOS had no effect on total serum cholesterol and HDL cholesterol levels.||Vulevic and others (2008)|
|V-GOS||Human||Increase lactobacilli but showed weak effect on bifidobacterial population. Decrease in β-glucoronidase and β-glucosidase and increase in azoreductase and nitroreductase.||Mcbain and Macfarlane (2001)|
|Milk fortified with bifidobacteria and GOS||Human||Supplementation with milk containing pre- and probiotics resulted in a significant reduction in incidence and prevalence of dysentery, a significant reduction in prevalence of severe illness, fever, and ear infections, no significance on diarrhea.||Sazawal and others (2004)|
|GOS (5% w/w)||Mice||GOS has no detectable effect on the intestine villus height but increased the total protein content of the small intestine mucosa by 2-fold. Sucrase activity was significantly increased in the intestinal mucosa recovered from animals fed the GOS diet without any detectable modification of lactase and phosphatase activities.||Leforestier and others (2009)|
|GOS||Human||GOS mixture containing mainly β1→3, as well as β1→4 and β1→6 linkages was found to be more bifidogenic than a GOS mixture containing mainly β1→4, as well as β1→6, after 1 wk of intake by healthy humans.||Depeint and others (2008)|
|B-GOS||Murine||GOS significantly reduced the colonization and pathology associated with S. typhimurium infection||Searle and others (2009)|
|GOS:FOS (9:1)||Human||Feeding with prebiotic mixture reduced the cumulative incidence of atopic dermatitis at 6 mo of age. Significantly higher number of fecal bifidobacteria compared with controls but there was no significant difference in lactobacilli counts.||Moro and others (2006)|
|GOS||Rat||The aberrant crypt multiplicity and the colorectal tumor incidence in rats fed an HGOS (20%, w/w) were significantly lower than those in rats fed an LGOS (5% w/w) diet.||Wijnands and others (2001)|
|GOS:FOS (9:1)||Human||GOS/FOS supplementation induced an antiallergic Ig profile in infants at high risk for allergic diseases||Nauta and others (2008)|
|GOS:FOS (9:1)||Human||The fecal concentration of sIgA was significantly higher in the infants that received an infant milk formula with added scGOS/lcFOS. Percentage of bifidobacteria was found to be higher and that of clostridium spp. was found to be lower in the scGOS/lcFOS group than in the control group.||Scholtens and others (2008)|
|GOS:FOS (9:1)||Human||No differences in total cholesterol and LDL cholesterol in infants receiving an infant formula with prebiotic mixture from infants receiving a control infant formula. Total cholesterol and LDL cholesterol levels were higher in breast-fed infants than in formula-fed infants.||Alliet and others (2007)|
|GOS with 4 probiotic organisms (L. rhamnosus GG, L. rhamnosus LC705, Bif. breve BB99, Propionibacterium freudenreichii ssp. shermanii JS)||Human||Significant reduction in IgE-associated diseases, eczema and atopic eczema.||Kukkonen and others (2007)|
|GOS:FOS||Human||Significant reduction in the plasma level of total IgE, IgG1, IgG2, and IgG3 (P = 0.0343) immunoglobulins. No significant effect on IgG4 was observed||Garssen and others (2007)|
|GOS||Rat||Increase in bifidobacterial numbers. No reduction in IBD inflammation.||Holma and others (2002)|
|B-GOS||Cell lines||Significant decrease in the attachment of enteropathogenic E. coli (EPEC) and S. enterica serovar Typhimurium to HT-29 epithelial cell line.||Tzortzis and others (2005)|
|B-GOS||Mice||Feeding of B-GOS prior to S. enterica serovar Typhimurium prevent the development of infection and the group receiving GOS mixture did not develop clinical symptoms of salmonellosis.||Searle and others (2009)|
|GOS, inulin, FOS, lactulose, or raffinose||Cell lines||GOS was shown to inhibit the adhesion of EPEC to Hep-2 and Caco-2 epithelial cell lines more effectively than inulin, FOS, lactulose, or raffinose.||Shoaf and others (2006)|
GOS and Intestinal Microflora
Bifidobacteria and lactobacilli are known to be potentially beneficial for health of hosts. Human milk is found to have various oligosaccharides, bifidogenic in nature. Studies have shown that bifidobacteria is the dominating species in the intestinal microflora of breast-fed infants, which differs from that of infants fed on cow's milk or other commercial formulated milk. Many harmful organisms like clostridia and enterococci and harmful chemicals like ammonia, amines, and phenols are found to be present in the intestine of formula-fed infants (Macfarlane and others 2008). The prevalence of bifidobacteria in breast-fed babies is thought to result from their abilities to utilize oligosaccharides in breast milk, including GOS. In a recent study with different strains of lactic acid bacteria, L. reuteri, L. fermentum, S. thermophilus, and L. mesenteroides subsp. cremoris, it was reported that these strains were not capable of utilizing complex HMOs but metabolized HMO components and GOSs (Schwab and Ganzle 2011). In some other studies, it was reported that feeding of a mixture of 10% long-chain FOS and 90% GOS to preterm infants resulted in an increase in intestinal bifidobacteria and lactobacilli, with a gut microbiota and fecal fermentation product composition more resembling that of breast-fed infants (Haarman and Knol 2005; Moro and Arslanoglu 2005).
The administration of a GOS mixture (3.6 g/d) containing mainly β1→3, as well as β1→4 and β1→6 linkages, proved to have a better bifidogenic effect than a GOS mixture (4.9 g/d) containing mainly β1→4, as well as β1→6, after 1 wk of intake by healthy humans (Depeint and others 2008). Both mixtures had mainly di- and trisaccharides. Both these GOS mixtures had low polymerization degree with DP ≥ 4 accounting for less than 12% and 19% of total saccharides, respectively.
GOS and Immune Modulation
The colonic microbiota is important for development and maturation of the immune system. Diet is one of the major factors that can influence the immune system in the gastrointestinal tract (Roller and others 2004) as well as intestinal microbial composition and metabolic product formation. Currently, there is increasing interest in the use of functional foods to modulate the gut immune system, with the aim of improving health and well-being. The gut contains a major part of the body's immune system, termed the gut-associated lymphoid tissue. Experimental-obtained data so far suggest that immune modulation of the gastrointestinal tract can occur through the use of functional foods such as prebiotics (Macfarlane and others 2008).
To date, few studies have been made on interactions between fermentable carbohydrates and the immune system, or whether they exert direct or indirect modulatory effects. Increased SCFA production, and increase in immunogenic bacteria such as lactobacilli and bifidobacteria are the 2 main methods by which prebiotics can exert their effects on the immune system. Fermentation of GOS (degradation of GOS by intestinal microflora) result in the production of butyrate, which serves as a fuel for colonic epithelial cells (Hopkins and Macfarlane 2003) stimulates apoptosis (Rowland 1998) suppress both cytokine-induced and constitutive expression of the transcription factor NF-κB in HT-29 cell lines (Inan and others 2000) and may be a protective factor in carcinogenesis (Scheppach and Weiler 2004). GOS fermentation in large intestine also produce propionate, which has been shown to be antiinflammatory with respect to colon cancer cells (Nurmi and others 2005).
A very recent study investigated the effect of GOS on microbiota composition and immune function (NK cells, phagocytosis, and cytokines) in healthy elderly volunteers. This study showed that GOS administration led to a significant decreases in the number of less beneficial bacteria (Bacteroides, Clostridium perfringens, Desulfovibrio spp., E. coli) and a significant increase in the number of beneficial bacteria especially bifidobacteria (Vulevic and others 2008). The study also found significant positive effect on immune response, evidenced by an improvement in NK cell activity and phagocytosis, increased secretion of the antiinflammatory cytokines, IL-10, and decreased secretion of pro-inflammatory cytokines (IL-6, IL-1β, and TNF-α).
There has been an increase in the incidence of allergic disease in developed countries over the last 2 decades, and studies have indicated that there may be evidence for a link between the colonic microbiota and allergy. Lower cell numbers of bifidobacteria with reduced adhesive properties have been found in the feces of allergic infants. These infants have IgE-mediated food allergies, and a Th2-biased immune response. Atopic dermatitis (AD) is one of the early signs of allergy in infancy, and can affect 10% to 25% of children in Western countries. In a study infants at risk of atopy were fed with prebiotic mixture composed of 90% GOS and 10% FOS and compared with a placebo group who were given maltodextrins (Moro and others 2006). Significantly higher numbers of bifidobacteria were found in the feces of the prebiotic group, compared to controls, but no increases in lactobacilli were observed. GOS when fed in combination with a mixture of 4 probiotic bacteria (L. rhamnosus GG, L. rhamnosus LC705, Bif. breve BB99, Propionibacterium freudenreichii ssp. shermanii JS) significantly reduce IgE-associated diseases, eczema, and atopic eczema (Kukkonen and others 2007).
In a double-blind randomized, placebo controlled, study on infants it was found that GOS/FOS supplementation induced an antiallergic immunoglobulin profile in infants at high risk for allergic diseases (Garssen and others 2007). Supplementation of GOS/FOS has lead to a significant reduction in the plasma level of total IgE (P = 0.007), IgG1 (P = 0.0054), IgG2 (P = 0.029), and IgG3 (P = 0.0343) immunoglobulins whereas no significant effect on IgG4 was observed. In one another study on infants, it was shown that GOS/FOS supplementation induces a beneficial antibody profile. GOS/FOS reduces the total Ig response and modulates the immune response towards CMP, while leaving the response to vaccination intact (Hoffen and others 2009).
Inflammatory Bowel Disease (IBD)
Inappropriate immune responses to the normal commensal gut microbiota sometimes result in inflammatory conditions like Ulcerative colitis and Crohn's disease (Cummings and others 2003). Reduced numbers of bifidobacteria and increased counts of other organisms such as E. coli and peptostreptococci have been found in fecal samples, and on the colonic mucosa in IBD (Macfarlane and others 2004). Normally, tolerance is mediated to the commensal microflora by release of the immunoregulatory cytokines IL-10 and TGF-b (Kelly and others 2005). Holma and others (2002) used the trinitrobenzene sulphonic acid (TNBS) model of colitis, in which rats were fed 4 g kg−1 body mass of GOS per day, 10 d before the induction of colitis, or dexamethasone at colitis induction, as a control. It was found that while there was an increase in bifidobacterial numbers in the animals, there was no reduction in inflammatory processes. Although this study do not show any effect of administration of GOS on IBD, this 1 animal model is not enough to draw any conclusions and more studies are needed to fully determine the potential of GOS in preventing or treating IBD.
FOS and inulin were used in several animal trials showing that administration of prebiotics is effective in lowering blood cholesterol level. However in vivo results are variable, with some studies reporting lowering effects and other no effects on blood cholesterol level. Thus far, GOS has been used in very few trials where serum cholesterol levels were investigated. In 1 trial, the effect of administration of 15 g/d of V-GOS, FOS and inulin were compared in healthy humans but no significant changes in serum lipids or glucose absorption were observed (Dokkum and others 1999). No significant difference in total cholesterol and LDL cholesterol was investigated in infants receiving an infant formula with GOS/lcFOS from infants receiving a control infant formula (Alliet and others 2007). Recently it was shown that 5.5 g of a GOS mixture administration to healthy elderly had no effect upon total serum cholesterol and HDL cholesterol levels (Vulevic and others 2008).
Antipathogenic Activity of GOS
Various studies have suggested that having prebiotics in diet protect the gut from infection and inflammation by inhibiting attachment and/or invasion of pathogenic bacteria or their toxins to colonic epithelium. This attachment is mediated by glycoconjugates on glycoproteins and lipids present on the microvillus membrane. Prebiotic GOS contain structures similar to those found on microvillus membrane that interfere with the bacterial receptor by binding to them and thus prevent bacterial attachment to colonic epithelium. α-linked GOS, present in human milk, are known to have antiadhesive properties and be capable of toxin neutralization (Newburg and others 2005, Morrow and others 2005). B-GOS contains an oligosaccharide in alpha anomeric configuration, and it was shown to significantly decrease the attachment of enteropathogenic E. coli (EPEC) and Salmonella enterica serovar Typhimurium to HT-29 epithelial cell line (Tzortzis and others 2005). The same GOS mixture was further studied in an oral challenge experiment, during which BalbC mice were fed with either a placebo or B-GOS prior to S. enterica serovar Typhimurium infection (Searle and others 2009). It was shown that the animals fed the GOS mixture did not develop clinical symptoms of salmonellosis, even though the pathogen could be recovered in the feces. Furthermore the histopathology and structure of the epithelium were completely protected and translocation of the pathogen to other organs was limited compared to placebo. In another study, GOS (oligomate) was shown to inhibit the adhesion of EPEC to Hep-2 and Caco-2 epithelial cell lines more effectively than inulin, FOS, lactulose, or raffinose (Shoaf and others 2006). However, the antiadhesive properties may be a result of GOS binding to pathogens and not a direct modulation of host immune system.
Factors Affecting GOS Yield
The amount of GOS produced in a reaction varies widely, and it depends on the reaction conditions mainly lactose concentration, enzyme source, glucose and galactose concentration, and temperature. Factors that increase the maximum concentration of GOS can be thought of as factors that increase the rate of GOS synthesis or decrease the rate of degradation (Gosling and others 2010). Data from the literature show that maximum GOS yield is largely influenced by initial lactose concentration. Since lactose solubility is relatively low at room temperature but manifestly increases with increasing temperature, high temperatures are generally desired (Roos 2009). Some studies have been focused on sourcing thermostable β-galactosidase because it seems that higher reaction temperature favor transgalactosylation reaction. β-galactosidase has been isolated from various thermostable microorganisms like S. solfataricus, P. furiosus, Thermus spp., T. caldophilus, C. saccharolyticus, T. maritima by various research groups. β-galactosidase from these organisms can be used for GOS production at a temperature around 80 °C and higher (Torres and others 2010). Various factors affecting GOS production are listed in Table 2.
Table 2–. Factors affecting GOS production.
|Lactose concentration 500 g/L, enzyme concentration 10 U/mL, temperature 45 °C and pH 7||GOS concentration, yield, and productivity were 83 g/L, 16.5%, and 27.6 g/L.h||Manera and others (2010)|
|Lactose (150, 250, and 350 mg/mL), temperature (40, 50, and 60 °C), pH (5.5, 6.5, and 7.5), enzyme (3, 6, and 9 U/mL)||Best reaction conditions for GOS production were 40 °C, pH 7.5, 250 mg/mL of lactose, 3 U/mL of enzyme, and 120 min.||Martinez-Villaluenga and others (2007)|
|Lactose contents 100, 200, and 300 g/L, pH 6.5, 40 GAU per gram lactose, temperature of 40 °C||Production of GOS-2 from 100 g/L, GOS-3 from 200 g/L, and GOS-4 from 300 g/L solution of lactose.||Adamczak and others (2009)|
|Lactose concentration 200 and 400 g/L||GOS production was found to be 21% (w/w) at lactose concentration of 200 g/L and 26% (w/w) at lactose concentration of 400 g/L. Addition of glucose and/or galactose reduced GOS production by 10% to 15%.||Albayrak and others (2002)|
|Lactose concentration 600 g/L, temperature 80 °C, pH 6||GOS production = 52.5% (w/w).||Park and others (2008)|
|Lactose concentration 500 g/L, temperature 40 °C, pH 4.5||52% (w/w) GOS was produced.||Neri and others (2009)|
|Lactose concentration 180 g/L, temperature 50 °C, pH 6||50% (w/w) GOS was produced.||Cho and others (2003)|
|Lactose concentration 205 g/L, temperature 37 °C, pH 6.5||GOS production was 38% (w/w).||Splechtna and others (2006)|
|Lactose concentration 400 g/L, temperature 45 °C, pH 6.8||GOS production was found to be 32.5% (w/w) of total sugar concentration||Hsu and others (2007)|
|Lactose concentration 400 g/L, temperature 40 °C, pH 7||The maximum amount of GOS produced was 24.8% (w/w).||Chockchaisawasdee and others (2005)|
|Initial lactose concentration of 500 g/L with temperature of 80 °C and pH of 6||19% of GOS was produced||Ji and others (2005)|
|Lactose concentration 450 to 500g/L||GOS yield values varied between 36% and 43%||Goulas and others (2007)|
|Lactose concentration 400 g/L||maximum productivity of GOS was 87 g/L·h||Zheng and others (2006)|
|Substrate: lactose in phosphate buffer (138 mmol/L), ultrafiltration permeate (115 mmol/L), recombined whey (136 mmol/L). Enzyme units: 0.15 to 15 U/mL for lactose in buffer, from 0.12 to 1.5 U/mL for ultrafiltration permeate and 1.5 U/mL for recombined whey||6.4 ± 0.4 mmol/L of GOS was obtained from lactose in buffer, 7.3 ± 0.1 mmol/L from ultrafiltration permeate, and 5.9 ± 0.1 mmol/L of GOS from recombined whey.||Hellerova and others (2009)|
Biotechnological Methods for Enhanced GOS Production
In past few years, a great deal of attention has been devoted to GOS synthesis, especially via enzymatic transglycosylation, since chemical synthesis of GOS is very tedious (Sears and others 2001). β-galactosidase from different microorganisms in both free and immobilized form have been employed for GOS synthesis (Neri and others 2008). Use of free enzymes and immobilized enzymes have their own limitations therefore an alternative strategy was proposed in which the enzyme is anchored on the cell surfaces of engineered microorganisms, such as E. coli and Saccharomyces cerevisiae (Lee and others 2003). In a recent study, a novel gene encoding transglycosylating β-galactosidase was cloned from Penicillium expansum F3 and was subsequently expressed on the cell surface of Saccharomyces cerevisiae EBY-100 by galactose induction. The β-galactosidase anchored yeast could directly utilize lactose to produce GOS, as well as the by-products glucose and a small quantity of galactose. The glucose was consumed by the yeast, and the galactose was used for β-galactosidase expression, thus greatly facilitating GOS synthesis (Li and others 2009).
A mutagenesis approach was applied to the β-galactosidase BgaB from Geobacillus stearothermophilus KVE39 to improve its enzymatic transglycosylation of lactose into oligosaccharides. (Placier and others 2009). The effects of the mutations on enzyme activity and kinetics were determined. Change of 1 arginine to lysine (R109K) increased the oligosaccharide yield compared to that for the wild-type BgaB. Subsequently, saturation mutagenesis at this position demonstrated that valine and tryptophan further increased the transglycosylation performance of BgaB. During the transglycosylation reaction with lactose of the evolved β-galactosidases, a major trisaccharide was formed. At the lactose concentration of 18% (wt/vol), this trisaccharide was obtained in yields of 11.5% (wt/wt) with GP21 (BgaB R109K), 21% with GP637.2 (BgaB R109V), and only 2% with the wild-type BgaB enzyme. GP643.3 (BgaB R109W) was shown to be the most efficient mutant, with a 3′-galactosyl-lactose production of 23%.
Recently novel β-galactosidase capable of glycosyl transfer was purified from a strain of Enterobacter cloacae B5 isolated from soil. This strain synthesizes GOS with a high yield of 55% from 275 g/L lactose at 50 °C for 12 h. A gene encoding the enzyme was cloned in E. coli and the recombinant enzyme was found to have similar transglycosylation activity to the natural enzyme (Lu and others 2009).
The different functional benefits from GOS are: high solubility, clean taste, high temperature and acid stability, and low glycemic index.
- 1GOS is 100 percent soluble in water and gives a transparent solution. GOS dissolves very well in milk and other dairy products and does not influence the viscosity of a beverage.
- 2The taste of GOS syrup is slightly sweet and neutral. GOS does not affect the taste or texture of your application and can, therefore, easily used in several applications and in several dosages.
- 3GOS syrup is very stable under low pH conditions. Storage at low pH will not degrade GOS and its content will remain stable over the shelf life. They are stable to pH 2 at 37 °C for several months making their application in nonrefrigerated fruit juice matrices possible.
- 4The presence of β-type linkages makes them resistant to high temperature in acidic medium. They remain unchanged after treatment at 160 °C for 10 min at neutral pH and after treatment at 120 °C for 10 min at a pH of 3 for 10 min at a pH of 2, offering potential for wide range of food applications.
- 5There are indications that low GI has a positive impact on diabetes, weight management, and cardiovascular diseases. They are not hydrolyzed by pancreatic enzymes and gastric juice passing the small intestine offering reduced glycemic index and a calorific value lower than 50% that of sucrose. Therefore they can be used in low-calorie diet foods and for consumption by peoples having diabetes.
Major Manufacturers of GOS
GOS have been manufactured and commercialized by very few companies all around the world. Some of them are reported to produce GOS for incorporation in to their own products. List of major manufacturers of GOS is given in Table 3.
Table 3–. Major manufacturer of GOS.
|Friesland Foods Domo (The Netherlands)||Vivinal GOS|
|Yakult Honsha (Japan)||Oligomate|
|GTC Nutrition (United States)||Purimune|
|Dairygold Food Ingredients (Ireland)||Dairygold GOS|
|First milk ingredients||Promovita|
|Nissin Sugar Manufacturing Company (Japan)||Cup-Oligo|
|Snow Brand Milk Products (Japan)||P7L|
|Clasado Ltd. (UK)||Bimuno|
GOS is an ideal ingredient to formulate into healthy products targeting specific groups such as infants, children, women, and the elderly. GOS can be easily incorporated in several applications such as: infant nutrition, growing up milk, dairy products, beverages, clinical nutrition, bakery, and pet food.
The rationale for supplementing an infant formula with prebiotics is to obtain a bifidogenic effect and the implied advantages of a “breast-fed-like” flora (Wauters and others 2005). GOS is an ideal ingredient for several types of infant formula. GOS are naturally present in mother's milk and provide several health benefits. GOS is already being used in many standards and premium infant formulas follow on formulas and growing up milks around the globe, GOS syrup is suited for wet processed infant formulas. A supplementation of low level of GOS (0.24 g/100 mL) in infant formula can improve stool frequency, decrease fecal pH, and stimulate intestinal bifidobacteria and lactobacilli as in those fed with human milk (Ben and others 2008).
Growing Up Milk
Growing up milk is for children 1 y or older. Its composition is different from breast milk or infant formula. Growing children have to obtain all the nutrients for their growth and development from food. GOS is already widely used in growing up milk. Because of its dairy origin and its use in infant formula, it is the most logical step to add in growing up milk.
GOS can easily be added to dairy applications such as milk yoghurts, buttermilk (Curda and others 2006), and dairy-based drinks due to its excellent solubility. In yoghurt, GOS can be added before (set or plain yoghurt) or after fermentation (sweetened or fruit yoghurt). Because of acid stability GOS can also be pre-blend with a fruit preparation. After the addition of GOS, structure of yoghurt was found to be little smoother and creamier. Additionally the yoghurt bacteria do not break GOS so it remains unmetabolized until it reaches the large intestine.
GOS can easily be incorporated in beverages like fruit juice, fruit drinks, breakfast drinks, and soft drinks because of its acid stability and property of forming clear solutions. It can easily be added together with other ingredients like concentrated fruit juices, compounds, or sugar syrup. GOS is heat and acid stable. No decrease of GOS is measured under low pH conditions and high temperatures. GOS is the perfect ingredient for use in acid drinks like soft or fruit based drinks and juices. Since it is very neutral and somewhat sweet in taste, the taste of beverages will not be influenced when GOS is added in them.
Clinical nutrition is a category of food and beverage products designed for individuals who are vulnerable due to illness. It is important to have the right diet that addresses their specific nutritional needs and prevents malnourishment for this group of people. GOS is an important ingredient to consider using in clinical nutrition because it stimulates the growth of bifidobacteria and provides several health benefits such as relief of constipation, support of natural defenses, and improved mineral absorption.
GOS can be used in development of bread and baked goods that are high in fiber, having low sugar content, and low calorie. GOS has an ideal combination of functional properties like low calorie and high-moisture retention capacity, which makes it an ideal component for baked products. Additionally it can provide several health benefits such as the growth of bifidobacteria, relief of constipation, support of natural defenses, and improved mineral absorption.
A strong immune system in animals starts with good intestinal health. A pet's immune system is affected by the bacteria that naturally occur in animal intestine. GOS helps a healthy intestinal environment by selectively stimulating the growth of “friendly” bacteria in the colon. There are a number of studies that have examined the effects of prebiotics in increasing the concentration of lactic acid bacteria such as lactobacilli or bifidobacteria or inhibition of pathogens; resulting in healthier pets with improved oral and fecal odor, skin, and coat health (http://www.vivinalgos.com).
The human gut is a complex ecosystem consisting of as many as 500 microbial species, and due to the stressful effects of various environmental and dietary factors, there is need to maintain or restore gut health. Incorporation of functional foods in diet has been proved to be a better approach to maintain a balance among the colonic microflora. Probiotics are already being used to prepare fermented dairy products that are becoming popular all around the globe. Nowadays, indigestible oligosaccharides are of particular interest and it seems that in the future prebiotic fibers will have a strong position in the nutraceuticals industry. Currently the main focus among prebiotics is on the production and use of GOS as a component of functional foods. GOS can be incorporated in different types of food products including mainly infant foods, confectionary, beverages, and clinical food. Various health benefits of GOS have been reported by different research groups. Absence of high-yielding techniques is the main limitation with GOS production methods. Additional research is needed to design and develop efficient methodologies to enhance the production of GOS in a cost effective manner. To overcome the limitation of poor yield, routine biological methods need to be rectified with modern biotechnological techniques.