Prebiotic Galactooligosaccharides: Enzymatic Synthesis, Characterization and Bioactive studies

Use of prebiotic oligosaccharides’ as nutraceutical food ingredients has
increased rapidly in the recent years due to their important physiological and
physicochemical attributes, which are known to improve the consumers’ health.
Galactooligosaccharides (GOS) have gained special attention towards human
nutrition owing to the presence of structurally related oligosaccharides, which bear a
semblance to the human milk oligosaccharides. GOS are oligomers of galactose with
either β(1'4), β(1'6), β(1'3) or β(1'2) linkages. They are produced from lactose through
transgalactosylation using β-galactosidase. In India, 270 million kg of whey/annum is
generated. It contains 16.2 million kg of valuable milk nutrients (lactose, protein, fat
and minerals) and could be an ideal source of lactose. Efforts towards the use of
whey have been made still, at present significant quantity is discarded as effluent.
The present study focuses on the GOS production using selected microbial β-
galactosidase from lactose as well as whey, from the point of view of value addition to
an industrial by-product.
Among the various microorganisms (78) evaluated, Lactobacillus plantarum
MCC2156 was selected for the production of β-galactosidase, based on its
transgalactosylating efficacy for GOS synthesis. Further, optimization of cultural and
nutritional parameters resulted in 1.7 fold increase in β-galactosidase production. The
β-galactosidase was purified to homogeneity using affinity chromatography, followed
by size exclusion chromatography and was found to be a 66 kDa monomeric protein
from SDS and native-PAGE analysis. In addition, Lactobacillus plantarum MCC2156
cells have been permeabilized for the use as whole cell biocatalyst.
Optimization of process parameter resulted in a maximum GOS yield of
34.1% (w/w) by 10 h when purified β-galactosidase was used for transgalactosylation
while 33.8% (w/w) GOS was obtained by 12 h in case of reaction with permeabilized
cells, at an optimum lactose concentration of 40% (w/v), pH 7.0 at 50oC. The
resultant GOS syrup contained (w/w): 38-45% monosaccharides (glucose and
galactose); 28-20% lactose; 33-35% GOS. The oligomer composition of GOS syrup
consists of 12- 14% GOS-2 (disaccharides), 15-17% GOS-3 (trisaccharides) and 3-
5% GOS-4 (tetrasaccharides). Besides, the comparative synthesis of GOS from
untreated whey and deproteinized whey was carried out under optimized conditions
using purified β-galactosidase which resulted in a maximum of 34.7 (w/w) and 34.4%
(w/w) GOS, respectively. Further, the microbial fermentation process was found to be
effective for the production of high purity GOS (91-93%) with a recovery of 81-84%.
Whey protein components such as lactoferrin, BSA, α- and β-casein, α- and β-
lactoglobulin were also retained, adding value to the GOS and high purity GOS
synthesised from whey.
In-vitro studies on prebiotic efficacy provided evidence towards the effective
fermentation of GOS by probiotic lactic acid bacteria, whereas GOS did not support
the growth of pathogenic bacteria. GOS was also found to have a profound influence
on the organic acids such as lactic, acetic and butyric acids production profile in
comparison with glucose. In addition, GOS significantly inhibited the adherence of E.
coli and B. cereus to the human epithelial (HEp-2) cell lines by acting as receptor
decoys. The results of in-vivo studies using female Wistar rats fed with GOS (10%,
w/w) supplemented diet for four weeks have clearly indicated in an effective
mobilization of calcium from Eleusine coracana (finger millet/ ragi) flour through gut
microbial fermentation.