UNIVERSIDAD DE GRANADA TESIS DOCTORAL Antonio Martínez Férez 2004

238 Pages·2007·4.31 MB·English

UNIVERSIDAD DE GRANADA

FACULTAD DE CIENCIAS
DEPARTAMENTO DE INGENIERÍA QUÍMICA

OBTENCIÓN DE OLIGOSACÁRIDOS DE LECHE DE
DIFERENTES ESPECIES POR TECNOLOGÍA DE
MEMBRANAS

TESIS DOCTORAL

Antonio Martínez Férez

2004
Editor: Editorial de la Universidad de Granada
Autor: Antonio Martínez Ferez
D.L.: Gr. 2930 - 2007
ISBN: 978-84-338-4711-9
TESIS DOCTORAL

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SUMMARY OF THE RESEARCH WORK

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OBTENTION OF OLIGOSACCHARIDES FROM MILK OF DIFFERENT SPECIES BY MEMBRANE
TECHNOLOGY

Breast milk is the ideal nourishment for newborn children, providing all the necessary nutrients for their
adequate growth and development, such as proteins, fats, carbohydrates, minerals and vitamins. In addition, breast milk
is the origin of a range of compounds with critical biochemical and physiological properties for the development and
growth of some newborn child's organs and tissues, as well as providing defence mechanisms against antigens and
pathogenic agents. Namely, breast milk contains hormones, growth factors, enzymes, proteins, bioactive peptides,
nucleotides, polyamines, oligosaccharides and long-chain polyunsaturated fatty acids. As a whole, these compounds
are called “milk trophic factors”. In particular, oligosaccharides are the third largest component of human milk, that
contains around 8-10 g/L and more than 130 different structures, which implies a complexity and variability of structures
not found in milk from other species.

The interest for oligosaccharides started about a hundred years ago after observing that these components
were most likely the factors responsible for the development of a bifidogenic flora in breastfed children. Nowadays, milk
oligosaccharides are thought to be beneficial for the human milk fed infant with regard to their prebiotic and anti-infective
properties. Indeed, there is currently a great interest in the role of these oligosaccharides as pathogen receptors. Since
they are synthesized by the same glycosyltransferases that join in the synthesis of glycoproteins and glycolipids from
the endothelium cells surface, it is possible the appearance of similarities among these glyco-compounds and milk
oligosaccharides. This fact stands up the hypothesis that asserts oligosaccharides may mimic cellular receptors for
pathogenic microorganisms, thus producing specific interactions between these oligosaccharides and pathogens and
acting as intestinal mucosa cell protectors.

Another important research topic is the influence of oligosaccharides on leukocyte-endothelial cell interactions.
Presently, there are therapeutic agents that block these interactions having dramatic effects on the progression of
inflammatory responses in numerous animal models. This effect could be obtained by applying the appropriate antibody
or using specific ligands (enzymatic synthesized carbohydrates) for selectins, a family of cell adhesion molecules, which
are directly implied on cell adhesion phenomena during inflammatory processes. It has been demonstrated that many
sialylated and fucosylated oligosaccharides from human milk have similar effects.

Besides all these properties previously raised, human milk oligosaccharides would be able to develop an
important function as suppliers of sialic acid, essential for the infant brain development, and galactose, the main
component of galactocerebrosides, the predominating glycolipid in myelin and cell membranes of neurones. Thus a
possible role of milk oligosaccharides is that they may ensure that the levels of these monosaccharides do not become
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limiting in the young mammal when its organism is not capable of providing all the amount needed, for example, during
the period of myelination and brain development.

On the basis of all these biological functions previously described for human milk oligosaccharides, which
reveal its important action in the development and growth of the newborn, the addition of these compounds to infant
formula would contribute a new step in the difficult challenge of imitate the human milk composition. On the other hand,
besides of the potential application in infant nutrition, milk oligosaccharides would find a market for supplementation of
specific functional foods for the elderly destined to the prevention of infections, and even in clinical and hospital
nutrition, for the treatment of patients with certain pathologies, contributing to a better and fast recovery, and definitely,
to improve their quality of life.

Unfortunately, in spite of the relevance of oligosaccharides for infant nutrition, most companies do not
supplement their formulas with them, since of the extreme difficulty of synthesis or isolation, in the case of finding a
natural source enriched in these biomolecules. In fact, no company supplements their infant formulas with
oligosaccharides similar to those found in human milk, and the ones doing so, just enrich with fructo- and/or galacto-
oligosaccharides, very simple structures which possess a prebiotic effect stimulating Bifidobacteria and Lactobacilli in
the gut, but where the other important functions described for human milk oligosaccharides are lacking. Other
components that currently are added with similar limitations are isomalto-oligosaccharides and oligosaccharides isolated
from soy.

In this sense, the research work described in this Thesis, belongs to the research activities that the Bioreactor
Group from the Department of Chemical Engineering of the University of Granada in collaboration with the Department
of Biomedicine from Puleva Biotech S.A. are carrying out for the development of new techniques to produce and purify
bioactive compounds. In summary, the objectives of this work are the following:

• Find a natural source of oligosaccharides similar to those found in breast milk for infant nutrition, among the
milk from the main farm mammals: cow, goat and sheep milk.

• Evaluation of its functional activity at a gastrointestinal level studying the influence of oligosaccharides on the
differentiation (alkaline-phosphatase, leucin-aminopeptidase and disacaridases activity), proliferation (taking
the PCNA as biomarker) and expression of mucins (MUC2, MUC4 y MUC5B) and “trefoil-factors” (TFF1 and
TFF3) of cultured intestinal cells (Caco-2 and HT29/MTX).

• Evaluation of its functional activity at an immunological level by means of the determination of the influence of
oligosaccharides on the monocytes adhesion to human umbilical vein endothelial cells (HUVEC) stimulated
with TNF-α under flow conditions.

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• Recovery of the oligosaccharide fraction by membrane technology. The aim pursued is to design and
implement an ultrafiltration process for the separation of the 90-95% of the oligosaccharide fraction from the
major proteins, caseins and whey proteins, without altering its biological properties neither its bioavailability.

• Dynamic modelling of the ultrafiltration process.

• Optimisation of the separation process to minimise the filtration area, which would determine the most
economic way of operation and would provide essential information for the scale up of the process.

The results obtained are exposed subsequently:

Selection of the natural source of oligosaccharides.

Goat milk is a good natural source of oligosaccharides regarding to both composition and concentration, and
the best among the commercially available milks examined (cow and sheep), containing a large array of acidic
oligosaccharides and N-acetyl-glucosaminyl-lactose together with galactosyl-lactose as major neutral structures. These
qualities have been proved by the following analytical techniques: gel filtration chromatography and high-pH anion-
exchange chromatography with pulsed amperometric detection (HPAEC-PAD) for the quantification and high-
performance thin layer chromatography (HP-TLC) and fast atom bombardment-mass spectrometry (FAB-MS) for the
characterization.

Although the content of oligosaccharides detected in goat milk is significantly lower compared to mature
human breast milk, however, structurally goat milk oligosaccharides and glycoconjugates are similar to those found in
human milk. Thus, it is reasonable to assume that the bioactivity of such molecules would also be similar and therefore
can be used in products for human nutrition.

The values of concentration of oligosaccharides and lactose detected in goat, cow, sheep and human milk are
shown in the following table:

Animal milk Oligosaccharides (g/L) Lactose (g/L)

Goat milk 0.25 – 0.30 45
Bovine milk 0.03 – 0.06 46

Ovine milk 0.02 – 0.04 48

Human milk 5 – 8 68

The analysis by HPAEC-PAD of each sample is shown subsequently:

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A. B.

3000 mV Neutral oligosaccharides Oligo1s5Aa ccicdhica rides 2 molecules of sialic acid 175 m V Neutral oligosaccLhaarcitdoesse oligosAaccicdhica rides 2 molecules of sialic acid
2500 8 150
2
PAD response12500000 1 4 5 9 11 13 14 16 20 PAD response 11702505 G L 6-S3L -SL
1000 3 6 7 10 12 17 18 19 50
500
25
00,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0 22,5 25,0 27,5 30,0 32,5 35,0 37,5 40,0 0,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0 22,5 25,0 27,5 30,0 32,5 35,0 37,5 40,0
Time (min) Time (min)

C. D.
300 m V Neutral Oligosaccharides oligosAaccicdhica rides 2 molecules of sialic acid mV Neutral oligosaccharides oligosAaccicdhica rides 2 molecules of sialic acid
PAD response221505000 LactosGeL 6-SL 3-SL PAD response754680000000000 1 2 1101 31 24 5 13 7 8 9 1 92 0
100 320000 6 1 4 15 1 6 1 178
5000,,00 22,,55 55,,00 77,,55 1100,,00 1122,,55 1155,,00 1177,,55 2200,,00 2222,,55 2255,,00 2277,,55 3300,,00 3322,,55 3355,,00 3377,,55 4400,,00 100 0,0 2,5 5,0 7,5 10,0 12,5 15,0 17,5 20,0 22,5 25,0 27,5 30,0 32,5 35,0 37,5 40,0
Time (min) Time (min)

Oligosaccharides profile by HPAEC-PAD of human (A), ovine (B), bovine (C) and caprine milk (D).

As can be seen in the previous figures, the chromatographic profile of goat milk is the most similar to human
milk, showing a greater number of peaks as compared to the chromatograms of sheep or cow’s milk, which show a
limited number of structures. Besides, the figures show that the sialylated fraction of goat milk is rich in structures
containing both one and two molecules of sialic acid, like human milk, while the sheep milk acidic fraction contains
mainly structures with two molecules of sialic acid and the cow’s milk acidic fraction shows molecules with just one
molecule of sialic acid.

The next figure shows the FAB-MS spectra from the goat milk oligosaccharide fraction, including some acidic
and neutral permethylated oligosaccharides. By this technique, we found 15 new oligosaccharide structures that have
never been reported so far to be present in goat milk: 4 are neutral and 11 are acidic oligosaccharides.

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4
868.6
3
100
838.5

1
80
) 477.2
Rel. intensity (%) Rel. Intensity (%rel intensity [%] 4600 6821 .4 10425.8 1 0672 1.81 7919 2.8211 92138.5780 96 .9 .801 41014. 1 1413231. 8 3
20 1463.9

600 800 1000 1200 1400 1600
mmm//zz/z

FAB-MS spectra of the goat milk oligosaccharide fraction.

In summary, the major oligosaccharides detected in goat milk by HPAEC-PAD and FAB-MS, their
abbreviations and concentrations are collected in the following table.

Abbreviation Name Concentration(g/L)

Acidic oligosaccharides

6-SL 6-Sialyl-lactose 0.07 – 0.08

3-SL 3-Sialyl-lactose 0.05 – 0.06

NGL N-glycolyl-neuraminyl-lactose 0.03 – 0.04

SLNH Sialyl-lacto-N-hexaose 0.001 – 0.005

DSL Di-sialyl-lactose 0.001 – 0.005

SNGL Sialyl-N-glycolyl-neuraminyl-lactose 0.001 – 0.005

SHL Sialyl-hexosyl-lactose Trace

NGHL N-glycolyl-neuraminyl-hexosyl-lactose Trace

SNGHL Sialyl-N-glycolyl-neuraminyl-hexosyl-lactose Trace

DSHL Di-sialyl-hexosyl-lactose Trace

DNGL Di-N-glycolyl-neuraminyl-lactose Trace

SDHL Sialyl-di-hexosyl-lactose Trace

DNGHL Di-N-glycolyl-neuraminyl-hexosyl-lactose Trace

NGLNH N-glycolyl-neuraminyl-lacto-N-hexaose Trace

Neutral oligosaccharides

GL Galactosyl-lactose 0.05 – 0.06

NAL N-acetyl-glucosaminyl-lactose 0.03 – 0.04
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LNH Lacto-N-hexaose 0.001 – 0.005

NALNH N-acetyl-glucosaminyl-lacto-N-hexaose 0.001 – 0.005

DNAL Di-N-acetyl-glucosaminyl-lactose 0.001 – 0.005

NAHL N-acetyl-glucosaminyl-hexosyl-lactose Trace

NADHL N-acetyl-glucosaminyl-di-hexosyl-lactose Trace

Total oligosaccharides 0.25 – 0.30

Evaluation of the functional activity of goat milk oligosaccharides at a gastrointestinal level.

Goat milk oligosaccharides induce the intestinal epithelium maturation, which involves a major resistance to
the adhesion of pathogens on the epithelial surface, and stimulate the cellular differentiation, but not the cellular
proliferation, since:

• the addition of purified goat milk oligosaccharides to the culture media of Caco-2 cells increases the majority
of the enzymatic activities assayed, like alkaline-phosphatase, lactase, maltase and sacarase, generating at
the same time a tendency of increase of the leucin-aminopeptisase activity.

• the supplementation of the culture media of Caco-2 and HT29/MTX cells with goat milk oligosaccharides, does
not produce any significant alteration in the levels of the proliferating cell nuclear antigen (PCNA), a biomarker
of proliferation widely used to study the effects of different nutrients present in the intestinal lumen on the
maintenance and proliferation of the colon epithelium.

Up to now, the interest for oligosaccharides has been focused on its role as pathogen receptors, protecting the
intestinal mucosa against the attack of pathogenic bacteria or as growth factors for Bifidobacterium and Lactobacillus,
but its implication on epithelial cells differentiation phenomena has never been addressed. In this sense, it has been
suggested that the glycosylation of the intestinal epithelium is a process regulated by the development, in which
probiotic bacteria and trophic factors present in the diet could have an important effect. This immaturity of the small
intestine could be a determinant factor in the colonization by microorganisms, being directly related to intestinal
infections in children. Therefore, the oligosaccharide fraction from human milk or any other ruminant, like goat milk
oligosaccharides, enriched in sialic acid, N-acetyl-glucosamine, glucose, galactose and N-glycolyl-neuraminic acid could
have an influence in the glycosylation patterns of epithelial cells toward a major level of differentiation.

On the other hand, the addition of purified goat milk oligosaccharides to the culture media has influence on the
genetic expression of mucoproteins and “trefoil-factors” synthesis in Caco-2 and HT29/MTX cells, producing a
significant and specific decrease in the expression of MUC2, MUC4 and MUC5B. Moreover, this supplementation
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