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).